Isolation and characterization of developmentally regulated sea urchin U2 snRNA genes

Genes encoding the U2 snRNA have been isolated from the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. Representatives of tandemly repeated gene sets have been isolated from both sea urchin species and a unique U2 gene has also been isolated from L. variegatus. The sequence of the U2 snRNA encoded by the tandemly repeated genes differs in two nucleotides between S. purpuratus and L. variegatus. The unique U2 gene from L. variegatus encodes the same U2 RNA as the tandemly repeated genes. There is a change in the U2 genes expressed between morula and pluteus embryos as judged by a change in the U2 RNA sequence in S. purpuratus embryos. The tandemly repeated genes were expressed at a higher rate in blastula than in gastrula stage relative to the single-copy gene, when the two genes were injected into sea urchin zygotes.     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.      

A developmental switch in sea urchin U1 RNA

The sequence of U1 RNA has been determined in the eggs and embryos of two sea urchins, Lytechinus variegatus and Strongylocentrotus purpuratus. In both species the sequence of the U1 RNA changes as the embryos progress through development. The sequence of the major U1 RNA in the eggs of the two species differs in two nucleotides, while the sequence of the U1 RNA present in the late embryos and somatic tissue is identical in the two species. The U1 RNA in eggs and early embryos is primarily derived from the tandemly repeated gene set, which is not expressed in somatic tissues.     

Pinocytosis of Ferritin from the Gut Lumen in Larvae of a Sea Star (Patiria miniata) and a Sea Urchin (Lytechinus pictus)

Pinocytosis of macromolecules from the gut lumen is demonstrated for the first time in larval stages of invertebrates. Developing sea stars ( Patiria miniata ) and sea urchins ( Lytechinus pictus ) were incubated in seawater containing ferritin, which was detected in cell organelles by transmission electron microscopy. Pinocytotic uptake of ferritin by gut cells of Patiria could be detected as soon as the larval mouth opened before the esophagus, stomach and intestine could be distinguished from one another; in contrast, no pinocytosis was detected at the comparable developmental stage (the prism larva) of Lytechinus . Pinocytosis was first detected in developing Lytechinus in pluteus larvae, especially in the stomach and intestine. In gut cells of both kinds of echinoderm larvae, the ferritin progressed rapidly from coated pits at the luminal cell membrane to secondary lysosomes (e.g. this progression took only about 10 min in stomach cells of Patiria larvae). Phagocytosis from the gut lumen was never observed after latex beads or starch granules were fed to larvae of Patiria and Lytechinus . Moreover, there was no evidence of pinocytosis of ferritin or phagocytosis of large particles by epidermal cells of larvae of either species.     

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.     

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.     

Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs

Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein.     

Selective expression of a sec1/munc18 member in sea urchin eggs and embryos

Regulated secretion is mediated by SNAREs (soluble NSF attachment receptors) and their regulators and effectors, which include the SM (sec1/munc18) family of proteins. Homologs of the SNAREs have been identified in sea urchins, associated with cortical granule exocytosis at fertilization, with membranes of the cleavage furrow, and in secretory cells later in development. To contribute to the understanding of regulated secretion in sea urchins we have cloned the single SM protein homolog from two species of sea urchin, Lytechinus variegatus and Strongylocentrotus purpuratus. In oocytes and eggs, we find that it localizes to the plasma membrane and the cortical region of the egg, consistent with a role in one of the steps leading to cortical granule exocytosis. The protein is also expressed throughout development, enriched in membranes of the cleavage furrow in early embryos, and in cells of the gut in advanced embryos. Furthermore, we find that sec1/munc18 co-localizes with its cognate binding partner syntaxin. Finally, our biochemical analysis shows that the protein associates with rab3 in high molecular weight complexes, suggesting that the exocytotic machinery functions as a multi-protein subunit to mediate regulated secretion in sea urchins. These results will be instrumental in the future to functionally test the SNARE regulators associated with multiple membrane fusion events.     

Ege A,a novel C2 domain containing protein,is essential for GPCR-mediated gene expression in dictyostelium

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.     

Influence of sperm and phytoplankton on spawning in the echinoid Lytechinus variegatus

The cues triggering large-scale broadcast-spawning events in marine invertebrates are not fully understood. Using the sea urchin Lytechinus variegatus, we tested the effectiveness of a variety of potential spawning cues in eliciting a spawning response. In the laboratory, during two consecutive spawning seasons, about 400 isolated sea urchins were exposed to phytoplankton, sperm, or eggs, singly or in combination. The likelihood of spawning, time to spawning, and spawning behavior were recorded for both sexes. Sperm was most successful at inducing spawning. No response to eggs was noted. Phytoplankton alone did not trigger spawning, but when a phytoplankton cue was followed by the addition of sperm, spawning behavior was induced, the time between addition of sperm and spawning was reduced, and the variance among individuals in the time of spawning initiation was reduced. Males spawned sooner in response to cues than females and rarely spawned spontaneously in phytoplankton or control treatments. A semilunar pattern in the sensitivity to spawning cues was noted. During time periods when sea urchins were less ripe, the ratio of spawning males to spawning females increased. Our results indicate that seasonal and lunar cycles, together with the presence of phytoplankton, increase the sensitivity of these sea urchins to spawning cues and the precision of their responses to conspecific sperm.     

Diameter-weight relationship and chromatic proportion of the sea urchin Lytechinus variegatus (Echinoidea: Toxopneustidae) in the islands of Margarita and Cubagua, Venezuela

In Margarita and Cabagua Islands, Venezuela, the sea urchin Lytechinus variegatus is consumed as food but few studies concern its biology. Between January 1999 and September 2000, 25 localities of Margarita and Cubagua islands we visited to determinate the relationship between diameter and weight of living urchins according to their coloration green or white. A total of 5 066 urchins (3 421 captured in Margarita and 1 645 in Cubagua) were measured. There is no significant difference in the weight-diameter relations of green or white sea urchins of both islands. The weight-diameter equation is P = 0.00246 D252. Utilizing a quadrant (0.5 x 0.5 m) monthly samples of urchins living in a 2 m2 of surface were taken to estimate the abundance of the specimens according their color (N = 6 948 urchins). The green urchins were more abundant than the white urchins, in a 3:2 proportion.     

Lack of age-associated telomere shortening in long- and short-lived species of sea urchins

The red sea urchin, Strongylocentrotus franciscanus, can live in excess of 100 years while the sea urchin Lytechinus variegatus has an estimated lifespan of only 3-4 years. In an effort to understand the molecular mechanism underlying the difference in their longevity we characterized telomere biology in these species of sea urchins. Telomerase activity was found throughout early stages of development in L. variegatus and is maintained in adult tissues of L. variegatus and S. franciscanus. Terminal restriction fragment analysis indicated a lack of age-associated telomere shortening. These data suggest that long- and short-lived sea urchins do not utilize telomerase repression as a mechanism to suppress neoplastic transformation.     

ULTRASTRUCTURE AND BIREFRINGENCE OF THE ISOLATED MITOTIC APPARATUS OF MARINE EGGS

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.     

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.     

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors leading to cannibalism in Lytechinus variegatus (Echinodermata: Echinoidia) held in intensive culture

Ecologically, sea urchins are an important species in marine habitats around the world. Economically, sea urchins are prized for their gonads (uni). With fisheries declining worldwide, intensive sea urchin culture has been proposed. For urchins in commercial culture, any factor that affects survivorship negatively should be addressed to maximize cost benefit. One potential obstacle to optimizing culture of sea urchins is cannibalism. Approximately 2000 adult and juvenile Lytechinus variegatus (1 g-45 g) were collected from Port Saint Joseph Peninsula State Park, FL between June and September 2009. Urchins were held in recirculating tanks at different sizes, densities, and feeding regimes for 4 weeks. Starvation and high density contributed to the highest level of cannibalism among small (12-21 g) urchins (percent cannibalism = 18.8%), whereas fed, high density conditions contributed to the highest level of cannibalism among large (32-37 g) urchins (percent cannibalism = 18.4%). These results suggest that (1) small urchins cannibalize at higher rates than large urchins, and (2) increased density is an important contributing factor leading to cannibalism. We quantified stress, defined as a decrease in production as a result of environmental conditions, by evaluating weight gain within each treatment and suggest that weight loss or minimal weight gain is an indicator of stress. We hypothesize increased stress caused by competitive interference can lead to increased cannibalism and decreased growth rates, even when food is not limiting. Ecologically, there are no reports of cannibalism of urchins in wild populations. Consequently, the role of cannibalism in regulating sea urchin community structure is not known. However, factors affecting cannibalism of L. variegatus in the laboratory may provide insight into the conditions that could result in cannibalism in wild populations. From an aquaculture perspective, it is important to determine those factors that contribute to the incidence of cannibalism in sea urchins so that the appropriate culture conditions can be maintained to reduce the incidence of cannibalism.     

Select light spectra affect gonad color in the sea urchin Lytechinus variegatus

It is suggested that gonad color in sea urchins depends upon the in vivo accumulation and metabolism of red and yellow carotenoid pigments. We hypothesized that differential light exposure could affect carotenoid deposition and, hence, gonad color in sea urchins. We therefore performed two experiments to determine whether light spectra affect the gonad color of Lytechinus variegatus. In the first experiment, urchins were fed a formulated feed supplemented with or without β-carotene and held beneath three lighting regimes designed to emit differing wavelengths of the visible spectrum. After 12 weeks, urchins were dissected and gonad color (CIE L*a*b*) was measured with a Pantone Capsure RM200. Actinic light significantly increased the value of a* (red) in gonad color. Color in the orange and yellow spectra in the gonads increased in individuals fed the β-carotene supplemented diet. In the second experiment, we cultured urchins for nine weeks under lamps specialized to emit UV radiation. All urchins in this experiment received diets supplemented with β-carotene. There was no significant difference in harvested gonad color between these treatments. These data suggest that light quality and dietary carotenoids affect carotenoid deposition in the gonads.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.