Novel origins of lineage founder cells in the direct-developing sea urchin Heliocidaris erythrogramma

The lineage and fate of each blastomere in the 32-cell embryo of the direct-developing sea urchin Heliocidaris erythrogramma have been traced by microinjection of tetramethylrhodamine-dextran. The results reveal substantive evolutionary modifications of the ancestral cell lineage pattern of indirect sea urchin development. Significant among these modifications are changes in the time and order of cell lineage segregation: vegetal ectodermal founder cells consistently arise earlier than during indirect development, while internal founder cells generally segregate later and in a different sequence. Modifications have also arisen in proportions of the embryo fated to become various cell types and larval structures. Ectodermal fates, particularly vestibular ectoderm, comprise a greater proportion of the total cellular volume in H. erythrogramma. Among internal cell types, coelom consumes more and endoderm less of the remaining cellular volume than during indirect sea urchin development. Evolutionary modifications are also apparent in the positional origin of larval cell types and structures in H. erythrogramma. These include an apparent tilt in the axis of prospective cell fate relative to the animal-vegetal axis as defined by cleavage planes. Together these evolutionary changes in the cell lineage of H. erythrogramma produce an accelerated loss of dorsoventral symmetry in cell fate relative to indirect development. The extent and diversity of rearrangements in its cell lineage indicate that the non-feeding larva of H. erythrogramma is a highly modified, novel form rather than a degenerate pluteus larva. These same modifications underscore the evolutionarily flexible relationship between cell lineage, gene expression, and larval morphology in sea urchin development.     

Nodal expression and heterochrony in the evolution of dorsal-ventral and left-right axes formation in the direct-developing sea urchin Heliocidaris erythrogramma

To understand the role of body axes in the evolution of larval form, we use the two sea urchins in the genus Heliocidaris, which have distinctly different larval morphologies. Heliocidaris tuberculata is an indirect-developing sea urchin, which forms a pluteus larva, whereas its sister species, Heliocidaris erythrogramma, exhibits direct development and forms a nonfeeding, ovoid larva. Changes along all three larval axes underlie the differences in larval form associated with each developmental mode. Nodal signaling has recently been implicated as important in establishing the dorsal-ventral (D-V) and left-right (L-R) axes in the indirect-developing sea urchin Paracentrotus lividus. However, because of changes in morphology and timing of morphogenetic events associated with the D-V and L-R axes, respectively, in H. erythrogramma, it was unclear whether nodal played the same roles during direct development. We show that the expression patterns and functions of nodal during H. erythrogramma development are similar to its roles in indirect-developing sea urchins in both D-V and L-R axes formation. However, there are profound changes in gene expression downstream of nodal signaling along the D-V axis and major heterochronies in the execution of the function of nodal along the L-R axis. These highly modified events are linked to the dramatic modifications of larval morphology that have occurred during the evolution of direct development in H. erythrogramma.     

Characterization of microsatellite loci for the Australian sea urchin Heliocidaris erythrogramma

We report 16 polymorphic microsatellite loci from Heliocidaris erythrogramma, a common sea urchin endemic to temperate Australian waters. These microsatellites were tested in a minimum of 30 individuals, which yielded between five and 14 alleles per locus. Expected heterozygosity ranged from 0.52 to 0.92 with four loci deviating from Hardy-Weinberg expectations. These markers are expected to be useful for experimental studies involving paternity analysis and for quantifying population structure in H. erythrogramma across its geographic range.     

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma

In the direct-developing sea urchin Heliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis in H. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

Genetic variation underlies temperature tolerance of embryos in the sea urchin Heliocidaris erythrogramma armigera

Ocean warming can alter natural selection on marine systems, and in many cases, the long‐term persistence of affected populations will depend on genetic adaptation. In this study, we assess the potential for adaptation in the sea urchin Heliocidaris erythrogramma armigera, an Australian endemic, that is experiencing unprecedented increases in ocean temperatures. We used a factorial breeding design to assess the level of heritable variation in larval hatching success at two temperatures. Fertilized eggs from each full‐sibling family were tested at 22 °C (current spawning temperature) and 25 °C (upper limit of predicted warming this century). Hatching success was significantly lower at higher temperatures, confirming that ocean warming is likely to exert selection on this life‐history stage. Our analyses revealed significant additive genetic variance and genotype‐by‐environment interactions underlying hatching success. Consistent with prior work, we detected significant nonadditive (sire‐by‐dam) variance in hatching success, but additionally found that these interactions were modified by temperature. Although these findings suggest the potential for genetic adaptation, any evolutionary responses are likely to be influenced (and possibly constrained) by complex genotype‐by‐environment and sire‐by‐dam interactions and will additionally depend on patterns of genetic covariation with other fitness traits.     

Spatial patterns of variation in color and spine shape in the sea urchin Heliocidaris erythrogramma

Abstract. Here we report on the first quantitative survey of morphological variation in the sea urchin Heliocidaris erythrogramma within Western Australia and distinguish between two subspecies found to co‐occur in this region. We surveyed urchins at multiple spatial scales along the Western Australian coastline to assess variation in dermis and spine color and, using landmark‐based geometric morphometrics, spine morphology. Both color and morphology proved to be useful for separating subspecies within Western Australia. There were four major color morphs: red dermis/violet spines (56%), red/violet‐green (23%), red/green (7%), and white/green (10%). Members of the first two color morphs had bulbous spines with wide, flattened tips, a morphology that is unique to Western Australia and characteristic of H. e. armigera, and members of the latter two consistently exhibited the narrow, pointed spines typical of specimens of H. e. erythrogramma, which has a broader distribution. In Western Australia, H. e. armigera was relatively abundant both within and among sites, but H. e. erythrogramma was found only in a few localized patches. Shifts in the relative abundance of these two subspecies occurred at fine spatial scales (<5 km), although environmental correlates of these transitions were unclear. Contrary to expectations, neither dermis color nor spine morphology varied with relative wave exposure: individuals with a red dermis or thickened spine morphology occurred at most sites regardless of exposure, and while white dermis and thinner spines only occurred at high‐exposure sites, these features were not common across the majority of exposed sites. Both color morph frequencies and spine morphology remained stable within sites over the 3‐year duration of this study. While the ecological significance of this morphological variation remains unclear, the consistency of the association between color and spine morphology, occurring across fine spatial scales, suggests that strong environmental or genetic factors are involved in maintaining morphological differentiation between these two subspecies.     

Co-option of an oral-aboral patterning mechanism to control left-right differentiation: the direct-developing sea urchin Heliocidaris erythrogramma is sinistralized, not ventralized, by NiCl2

Larval dorsoventral (DV) and left-right (LR) axial patterning unfold progressively in sea urchin development, leading to commitment of the major embryonic regions by the gastrula stage. The direct-developing sea urchin Heliocidaris erythrogramma has lost oral-aboral differentiation along the DV axis but has accelerated vestibular ectoderm development on the left side. NiCl(2) radializes indirect-developing sea urchins by shifting cells toward a ventral fate (oral ectoderm). We treated embryos of H. erythrogramma and the indirect-developing H. tuberculata with NiCl(2). H. tuberculata was ventralized exactly like other indirect developers, establishing that basic patterning mechanisms are conserved in this genus. H. erythrogramma was also radialized; timing, dosage response, and some morphological features were similar to those in other sea urchins. Ectodermal explant and recombination experiments demonstrate that the effect of nickel is autonomous to the ectoderm, another feature in common with indirect developers. However, H. erythrogramma is distinctly sinistralized rather than ventralized, its cells shifting toward a left-side fate (vestibular ectoderm). This geometric contrast in the midst of pervasive functional similarity suggests that nickel-sensitive processes in H. erythrogramma axial patterning, homologous to those in indirect developers, have been redeployed, and hence co-opted, from their ancestral role in DV axis determination to a new role in LR axis determination. We discuss DV and LR axial patterning and their evolutionary transformation.     

Male-by-female interactions influence fertilization success and mediate the benefits of polyandry in the sea urchin Heliocidaris erythrogramma

Numerous studies have reported that females benefit from mating with multiple males (polyandry) by minimizing the probability of fertilization by genetically incompatible sperm. Few, however, have directly attributed variation in female reproductive success to the fertilizing capacity of sperm. In this study we report on two experiments that investigated the benefits of polyandry and the interacting effects of males and females at fertilization in the free-spawning Australian sea urchin Heliocidaris erythrogramma. In the first experiment we used a paired (split clutch) experimental design and compared fertilization rates within female egg clutches under polyandry (eggs exposed to the sperm from two males simultaneously) and monandry (eggs from the same female exposed to sperm from each of the same two males separately). Our analysis revealed a significant fertilization benefit of polyandry and strong interacting effects of males and females at fertilization. Further analysis of these data strongly suggested that the higher rates of fertilization in the polyandry treatment were due to an overrepresentation of fertilizations due to the most compatible male. To further explore the interacting effects of males and females at fertilization we performed a second factorial experiment in which four males were crossed with two females (in all eight combinations). In addition to confirming that fertilization success is influenced by male x female interactions, this latter experiment revealed that both sexes contributed significant variance to the observed patterns of fertilization. Taken together, these findings highlight the importance of male x female interactions at fertilization and suggest that polyandry will enable females to reduce the cost of fertilization by incompatible gametes.     

The "lecithotrophic" sea urchin Heliocidaris erythrogramma lacks typical yolk platelets and yolk glycoproteins

The sea urchin Heliocidaris tuberculata undergoes typical development, forming an echinoid pluteus larva, whereas H. erythrogramma undergoes direct development via a highly modified, nonfeeding larva. Using a polyclonal antibody prepared against yolk glycoproteins from the typical developer Stronglyocentrotus purpuratus, we found that H. tuberculata contains cross-reactive proteins in abundance, but H. erythrogramma does not. In addition, we used immunoelectron microscopy to demonstrate that unfertilized eggs of H. tuberculata contain yolk platelets, but those of H. erythrogramma do not.     

The dorsoventral axis is specified prior to first cleavage in the direct developing sea urchin Heliocidaris erythrogramma

Previous fate mapping studies as well as the culture of isolated blastomeres have revealed that the dorsoventral axis is specified as early as the 2-cell stage in the embryos of the direct developing echinoid, Heliocidaris erythrogramma. Normally, the first cleavage plane includes the animal-vegetal axis and bisects the embryo between future dorsal and ventral halves. Experiments were performed to establish whether the dorsoventral axis is set up prior to the first cleavage division in H. erythrogramma. Eggs were elongated and fertilized in silicone tubes of a small diameter in order to orient the cleavage spindle and thus the first plane of cell division. Following first cleavage, one of the two resulting blastomeres was then microinjected with a fluorescent cell lineage tracer dye. Fate maps were made after culturing these embryos to larval stages. The results indicate that the first cleavage division can be made to occur at virtually any angle relative to the animal-vegetal and dorsoventral axes. Therefore, the dorsoventral axis is specified prior to first cleavage. We argue that this axis resides in the unfertilized oocyte rather than being set up as a consequence of fertilization.     

Larval settlement of the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the surface of coralline algae

Bacterial biofilms are increasingly seen as important for the successful settlement of marine invertebrate larvae. Here we tested the effects of biofilms on settlement of the sea urchin Heliocidaris erythrogramma. Larvae settled on many surfaces including various algal species, rocks, sand and shells. Settlement was reduced by autoclaving rocks and algae, and by treatment of algae with antibiotics. These results, and molecular and culture-based analyses, suggested that the bacterial community on plants was important for settlement. To test this, approximately 250 strains of bacteria were isolated from coralline algae, and larvae were exposed to single-strain biofilms. Many induced rates of settlement comparable to coralline algae. The genus Pseudoalteromonas dominated these highly inductive strains, with representatives from Vibrio, Shewanella, Photobacterium and Pseudomonas also responsible for a high settlement response. The settlement response to different bacteria was species specific, as low inducers were also dominated by species in the genera Pseudoalteromonas and Vibrio. We also, for the first time, assessed settlement of larvae in response to characterised, monospecific biofilms in the field. Larvae metamorphosed in higher numbers on an inducing biofilm, Pseudoalteromonas luteoviolacea, than on either a low-inducing biofilm, Pseudoalteromonas rubra, or an unfilmed control. We conclude that the bacterial community on the surface of coralline algae is important as a settlement cue for H. erythrogramma larvae. This study is also an example of the emerging integration of molecular microbiology and more traditional marine eukaryote ecology.     

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.     

Evolutionary dissociation between cleavage, cell lineage and embryonic axes in sea urchin embryos.

Using vital dye staining and the microinjection of fluorescent cell lineage-autonomous tracers, the relationship between the first cleavage plane and the prospective larval dorsoventral axis was examined in several sea urchin species, including: Strongylocentrotus purpuratus, S. droebachiensis, Lytechinus pictus, Clypeaster rosaceus, Heliocidaris tuberculata and H. erythrogramma. The results indicate that there is no single relationship between the early cleavage pattern and the dorsoventral axis for all sea urchins; however, specific relationships exist for individual species. In S. purpuratus the first cleavage plane occurs at an angle 45 degrees clockwise with respect to the prospective dorsoventral axis in most cases, as viewed from the animal pole. On the other hand, in S. droebachiensis, L. pictus and H. tuberculata, the first cleavage plane generally corresponds with the plane of bilateral symmetry. There does not appear to be a predominant relationship between the first cleavage plane and the dorsoventral axis in C. rosaceus. In the direct-developing sea urchin H. erythrogramma the first cleavage plane bisects the dorsoventral axis through the frontal plane. Clearly, evolutionary differences have arisen in the relationship between cleavage pattern and developmental axes. Therefore, the mechanism of cell determination is not necessarily tied to any particular pattern of cell cleavage, but to an underlying framework of axial systems resident within sea urchin eggs and embryos.     

Fine‐scale patterns of genetic divergence within and between morphologically variable subspecies of the sea urchin Heliocidaris erythrogramma (Echinometridae)

The spatial scale over which genetic divergences occur between populations and the extent that they are paralleled by morphological differences can vary greatly among marine species. In the present study, we use a hierarchical spatial design to investigate genetic structure in Heliocidaris erythrogramma occurring on near shore limestone reefs in Western Australia. These reefs are inhabited by two distinct subspecies: the thick‐spined Heliocidaris erythrogramma armigera and the thin‐spined Heliocidaris erythrogramma erythrogramma, each of which also have distinct colour patterns. In addition to pronounced morphological variation, H. erythrogramma exhibits a relatively short (3–4 days) planktonic phase before settlement and metamorphosis, which limits their capacity for dispersal. We used microsatellite markers to determine whether patterns of genetic structure were influenced more by morphological or life history limitations to dispersal. Both individual and population‐level analyses found significant genetic differentiation between subspecies, which was independent of geographical distance. Genetic diversity was considerably lower within H. e. erythrogramma than within H. e. armigera and genetic divergence was four‐fold greater between subspecies than among populations within subspecies. This pattern was consistent even at fine spatial scales (< 5 km). We did detect some evidence of gene flow between the subspecies; however, it appears to be highly restricted. Within subspecies, genetic structure was more clearly driven by dispersal capacity, although weak patterns of isolation‐by‐distance suggest that there may be other factors limiting gene exchange between populations. Our results show that spatial patterns of genetic structure in Western Australian H. erythrogramma is influenced by a range of factors but is primarily correlated with the distribution of morphologically distinct subspecies. This suggests the presence of reproductive barriers to gene exchange between them and demonstrates that morphological variation can be a good predictor of genetic divergence. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 578–592.     

Effects of ocean warming and acidification on survival,growth and skeletal development in the early benthic juvenile sea urchin (Heliocidaris erythrogramma)

Co‐occurring ocean warming, acidification and reduced carbonate mineral saturation have significant impacts on marine biota, especially calcifying organisms. The effects of these stressors on development and calcification in newly metamorphosed juveniles (ca. 0.5 mm test diameter) of the intertidal sea urchin Heliocidaris erythrogramma, an ecologically important species in temperate Australia, were investigated in context with present and projected future conditions. Habitat temperature and pH/pCO₂ were documented to place experiments in a biologically and ecologically relevant context. These parameters fluctuated diurnally up to 10 °C and 0.45 pH units. The juveniles were exposed to three temperature (21, 23 and 25 °C) and four pH (8.1, 7.8, 7.6 and 7.4) treatments in all combinations, representing ambient sea surface conditions (21 °C, pH 8.1; pCO₂ 397; Ω_Ca 4.7; Ω_Ar 3.1), near‐future projected change (+2–4 °C, ?0.3–0.5 pH units; pCO₂ 400–1820; Ω_Ca 5.0–1.6; Ω_Ar 3.3–1.1), and extreme conditions experienced at low tide (+4 °C, ?0.3–0.7 pH units; pCO₂ 2850–2967; Ω_Ca 1.1–1.0; Ω_Ar 0.7–0.6). The lowest pH treatment (pH 7.4) was used to assess tolerance levels. Juvenile survival and test growth were resilient to current and near‐future warming and acidification. Spine development, however, was negatively affected by near‐future increased temperature (+2–4 °C) and extreme acidification (pH 7.4), with a complex interaction between stressors. Near‐future warming was the more significant stressor. Spine tips were dissolved in the pH 7.4 treatments. Adaptation to fluctuating temperature‐pH conditions in the intertidal may convey resilience to juvenile H. erythrogramma to changing ocean conditions, however, ocean warming and acidification may shift baseline intertidal temperature and pH/pCO₂ to levels that exceed tolerance limits.     

Morphogenetic mechanisms of coelom formation in the direct-developing sea urchin <Emphasis Type="Italic">Heliocidaris erythrogramma</Emphasis>

Indirect development via a feeding pluteus larva represents the ancestral mode of sea urchin development. However, some sea urchin species exhibit a derived form of development, called direct development, in which features of the feeding larva are replaced by accelerated development of the adult. A major difference between these two developmental modes is the timing of the formation of the left coelom and initiation of adult development. These processes occur much earlier in developmental and absolute time in direct developers and may be underlain by changes in morphogenetic processes. In this study, we explore whether differences in the cellular mechanisms responsible for the development of the left coelom and adult structures are associated with the change in the timing of their formation in the direct-developing sea urchin Heliocidaris erythrogramma. We present evidence that left coelom formation in H. erythrogramma, which differs in major aspects of coelom formation in indirect developers, is not a result of cell division. Further, we demonstrate that subsequent development of adult structures requires cell division.