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Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization.

Sulfated polysaccharides from egg jelly are the molecules responsible for inducing the sperm acrosome reaction in sea urchins. This is an obligatory event for sperm binding to, and fusion with, the egg. The sulfated polysaccharides from sea urchins have simple, well defined repeating structures, and each species represents a particular pattern of sulfate substitution. Here, we examined the egg jellies of the sea urchin sibling species Strongylocentrotus droebachiensis and Strongylocentrotus pallidus. Surprisingly, females of S. droebachiensis possess eggs containing one of two possible sulfated fucans, which differ in the extent of their 2-O-sulfation. Sulfated fucan I is mostly composed of a regular sequence of four residues ([4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-1-->4-alpha-l-Fucp-1]n), whereas sulfated fucan II is a homopolymer of 4-alpha-l-Fucp-2(OSO3)-1 units. Females of S. pallidus contain a single sulfated fucan with the following repeating structure: [3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1]n. The egg jellies of these two species of sea urchins induce the acrosome reaction in homologous (but not heterologous) sperm. Therefore, the fine structure of the sulfated alpha-fucans from the egg jellies of S. pallidus and S. droebachiensis, which differ in their sulfation patterns and in the position of their glycosidic linkages, ensures species specificity of the sperm acrosome reaction and prevents interspecies crosses. In addition, our observations allow a clear appreciation of the common structural features among the sulfated polysaccharides from sea urchin egg jelly and help to identify structures that confer finer species specificity of recognition in the acrosome reaction.

Acrosome Reaction↗

Structure, expression, and transcriptional regulation of the Strongylocentrotus franciscanus spec gene family encoding intracellular calcium-binding proteins.

The mechanisms by which gene expression patterns emerge during evolution are poorly understood. The sea urchin spec genes offer a useful means to investigate evolutionary mechanisms. Genes of the spec family from Strongylocentrotus purpuratus and Lytechinus pictus have identical patterns of aboral ectoderm-specific expression but exhibit species-specific differences in copy number, genomic structure, temporal expression, and cis-regulatory architecture. Here, we identify spec genes from a phylogenetic intermediate, Strongylocentrotus franciscanus, to gain insight into the evolution of the spec gene family and its transcriptional regulation. We identified two spec genes in the S. franciscanus genome, sfspec1a and sfspec1b, that were orthologous to spec1 from S. purpuratus. sfspec1b transcripts began to accumulate at the blastula stage and became progressively more abundant; this was reminiscent of spec expression in L. pictus but different from that in S. purpuratus. As expected, sfspec1b expression was restricted to aboral ectoderm cells. The six-exon structure of the sfspec1b genomic locus was identical to that of the S. purpuratus spec genes and was bounded by two repeat-spacer-repeat (RSR) repetitive sequence elements, which are conserved features of S. purpuratus spec genes and function as transcriptional enhancers. The enhancer activity of the sfspec1b RSRs was comparable to that of their S. purpuratus counterparts, although the placement and orientation of crucial cis-regulatory elements within the RSRs differed. We discovered a spec gene in S. franciscanus that was only distantly related to other spec genes but was highly conserved in S. purpuratus. Unexpectedly, this gene was expressed exclusively in endoderm lineages. Our results show that the evolution of spec cis-regulatory elements is highly dynamic and that substantial alterations can occur when maintaining or grossly modifying gene expression patterns.

Amino Acid Sequence↗

Structure and organization of the CyIII actin gene subfamily of the sea urchin, Strongylocentrotus purpuratus.

We describe here the organization of the CyIII subfamily of cytoskeletal actin genes in the sea urchin Strongylocentrotus purpuratus. The functional genes CyIIIa and CyIIIb are linked at a 6 X 10(3)-base distance. Gene CyIIIc appears to be a pseudogene that lacks 5' exons and displays unselected mutational changes. Gene CyIIIa codes for a protein that differs at only nine out of 376 residues from that coded by another cytoskeletal actin gene, CyI. However, five of these nine changes occur within an 11-amino acid region that could represent a functional specialization of the CyIIIa actin protein. The CyIIIa gene possesses three introns, located, respectively, 25 nucleotides upstream from the translation start site, between the codons for amino acids 121 and 122, and within the codon for amino acid 204. These intron positions have also been observed in other cytoskeletal sea urchin actin genes. Comparison of both intron and 3'-terminal sequences shows that the CyIIIa and CyIIIb genes are closely related, while no homology in these untranslated sequences is observed between the CyIII genes and the other cytoskeletal actin genes of the S. purpuratus genome. The CyIII genes probably arose by duplication events at least 40 X 10(6) years ago, prior to radiation of the genus Strongylocentrotus. Consideration of the biological role of the embryo and larval aboral ectoderm cells to which CyIIIa and CyIIIb transcripts are confined suggests that these actins might contribute to cytoskeletal elements that endow the larval body wall with its rigid structure.

Actins↗

Cholinoreceptors of the dentis retractor muscle of the sea urchin Strongylocentrotus intermedius.

Neostigmine increases and cholinergic blocking agents block contractions of Strongylocentrotus intermedius dentis retractor muscle caused by either acetylcholine or indirect stimulation. Both innervated and non-innervated parts of the muscle are sensitive to low acetylcholine concentration suggesting that there are both synaptic and extra-synaptic cholinoreceptors. Nicotinomimetics are more potent than muscarinomimetics. Both d-tubocurarine and atropine are weak blocking agents as compared to pentadecamethylene-bis-trimethylammonium or hexadecamethylene-bis-trimethylammonium, Kd being 1 X 10(-4) to 5 X 10(-5) and 5 X 10(-7) to 4 X 10(-7) M respectively. Disulphide bonds and carboxylic (or phosphate) groups were revealed in dentis retractor muscle cholinoreceptors using drugs modifying receptor structure. Cholinoreceptors of both nicotinic and muscarinic type were revealed in oesophagus longitudinal muscles of Strongylocentrotus intermedius.

Animals↗

Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.

Amino Acid Sequence↗

Gene expression patterns in the intestines of sea urchins (Strongylocentrotus intermedius) under prolonged high-salinity stress.

The effective development of high-salinity aquaculture for the sea urchin Strongylocentrotus intermedius depends on understanding its molecular mechanisms. Therefore, we conducted a 60-day experiment to investigate the effects of prolonged high-salinity stress on the survival, growth, amino acid levels, antioxidant enzyme activity, and gene expression of S. intermedius. The experiment involved the preparation of two groups: one with a salinity of 32 (group S32) and another with 36 (group S36). The results showed that the survival rate of S. intermedius in group S36 was 80%&#xa0;&#xb1;&#xa0;6.7%, while the weight gain rate was only 61.58%&#xa0;&#xb1;&#xa0;1.92%. Both parameters were significantly lower than those in group S32 (P&#xa0;<&#xa0;0.05). In addition, the GSH, Cys, and Glu expression in S. intermedius was significantly higher than that observed in group S32 (P&#xa0;<&#xa0;0.05). The transcriptomic results showed that, when comparing groups S32 and S36, 179 differentially expressed genes were identified. These genes were predominantly enriched in pathways related to metabolism and amino acid biosynthesis. We highlight the genes CGL, EAAT3, AMY, and NADH, which are associated with the energy metabolism, cysteine transport, and amino acid biosynthesis of S. intermedius. We speculated that S. intermedius exposed to high salinity enhances energy metabolism, as well as Cys synthesis and transport, to mitigate oxidative stress. This study provides a theoretical reference for the healthy aquaculture of S. intermedius in high-salinity environments.

Animals↗

Physiological and molecular responses of coelomocytes to low- to mid-frequency acoustic exposure in the sea urchin Strongylocentrotus intermedius.

Underwater noise is a widespread environmental pollutant in marine ecosystems, yet the effects of low- to mid-frequency acoustic exposure on immune physiology and molecular responses in echinoderms remain unclear. In this study, the sea urchin Strongylocentrotus intermedius was exposed to continuous pure-tone acoustic stimulation at 80, 125, 250, 500, 750, and 1000&#xa0;Hz for 3&#xa0;h. Results showed that acoustic exposure significantly affected redox homeostasis, energy metabolism, and immune function in S. intermedius coelomocytes: the antioxidant system and glutathione redox balance were altered, as indicated by increased superoxide dismutase (SOD) and catalase (CAT) activities, elevated reduced glutathione (GSH) content, and a higher GSH/GSSG ratio; glycolysis-related enzyme activities were enhanced, with increased pyruvate kinase (PK) activity under 125-500&#xa0;Hz exposure and elevated hexokinase (HK) activity at 250&#xa0;Hz; and immune function was impaired, as shown by increased coelomocyte mortality, reduced phagocytic activity, and inhibited acid phosphatase (ACP) and alkaline phosphatase (AKP) activities, whereas respiratory burst activity showed no significant change. Among all treatments, 250&#xa0;Hz induced the most pronounced physiological responses. Transcriptomic analysis of coelomocytes from the 250&#xa0;Hz group identified 663 differentially expressed genes, including 537 upregulated and 126 downregulated genes, mainly enriched in pathways related to apoptosis, phagosome, lysosome, glutathione metabolism, arachidonic acid metabolism, and carbohydrate metabolism. These findings indicate that low- to mid-frequency acoustic exposure can act as a physiological and molecular stressor to S. intermedius coelomocytes by affecting redox homeostasis, enhancing energy metabolism, and suppressing immune effector processes, with 250&#xa0;Hz showing the strongest effect under the present exposure conditions. This study provides experimental evidence for evaluating the potential biological effects of low- to mid-frequency acoustic exposure on benthic echinoderms.

Animals↗

Morphological changes and transcriptomic insights into skeletal development of embryos and larvae of the sea urchin Strongylocentrotus intermedius.

To explore morphological features and molecular dynamics underlying skeletogenesis in the sea urchin Strongylocentrotus intermedius, we conducted combined morphological observation and comparative transcriptome analyses across representative embryonic and larval developmental stages. Morphological results showed that triradiate spicules first emerged at the gastrula stage. The 8-arm pluteus stage was identified as a key phase for skeletal remodeling, during which new three-radiate crystals transformed into complex stereoscopic ossicles including tube feet, spines and test plates. Transcriptomic data indicated that most differentially expressed genes (DEGs) were downregulated from the blastula to gastrula. The altered expression of basal metabolic genes and extracellular matrix genes including Colp2&#x3b1; and calm may be correlated with the linear mineralization of early spicules, which potentially reflects an energy adjustment pattern in developing larvae. During the transition from 6-arm to 8-arm pluteus, expression changes of calmodulin-like, Colp2&#x3b1; and SISin18G001660 suggest potential associations with regional calcium deposition and modifications of skeletal matrix properties. This work systematically characterizes morphological traits and transcriptional dynamics of skeletogenesis in S. intermedius. Its early spiculogenesis follows the conserved developmental pattern of echinoderms, while massive formation of stereoscopic ossicles occurs at the 8-arm pluteus stage. Stage-specific transcriptional changes across key larval skeletogenic stages are uncovered, offering transcriptomic resources for functional verification of skeletal regulatory genes.

Animals↗

Sphedgehog is expressed by pigment cell precursors during early gastrulation in Strongylocentrotus purpuratus.

We have sequenced the Sphedgehog (Sphh) gene from the sea urchin Strongylocentrotus purpuratus. Sphh transcripts are detected first at the mesenchyme blastula stage, and they accumulate throughout early embryogenesis. The Sphh protein is produced by precursor pigment cells during early and midgastrulation. NiCl2 inhibits pigment cell differentiation in sea urchins. Here, we show that, in S. purpuratus, nickel affects a process(es) between 17 and 24 hr of development, corresponding to the time period when Sphh mRNA is first detected. However, nickel treatment does not alter the early expression of Sphh.

Amino Acid Sequence↗

Strongylocentrotus purpuratus transcription factor GATA-E binds to and represses transcription at an Otx-Goosecoid cis-regulatory element within the aboral ectoderm-specific spec2a enhancer.

During Strongylocentrotus purpuratus embryogenesis, aboral ectoderm-specific expression of spec2a relies on an upstream enhancer that confers its spatial specificity largely through repression. The purpose of this study was to determine how spec2a expression is repressed in endoderm and oral ectoderm territories. A 78-base pair DNA sequence within the enhancer contains five tightly spaced cis-regulatory elements including proximal (TAATCT) and distal (TAATCC) elements that bind to both SpOtx, a broadly distributed transcriptional activator, and SpGoosecoid (SpGsc), an oral ectoderm-restricted transcriptional repressor. We show here that these two seemingly redundant Otx/Gsc elements have distinct functions. The proximal element bound to SpGATA-E, an endomesoderm-specific transcription factor. Treatment with SpGATA-E and SpGsc morpholino antisense oligonucleotides (MASOs) resulted in enhanced transcriptional activity from the proximal element, suggesting that both factors functioned as repressors at this site. SpGATA-E MASO-treated embryos failed to express ectoderm markers, indicating a role for SpGATA-E in ectoderm differentiation. The spec2a proximal element was distinct from the corresponding element in the related spec1 enhancer, and swaps between spec1 and spec2a cis-regulatory elements indicated, that for optimal repression, the proximal element had to interact with a nearby CCAAT-binding factor element. Our results show that the recently evolved proximal element contributes to the repression of spec2a in endomesoderm and oral ectoderm territories.

Amino Acid Sequence↗

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus.

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.

Analysis of Variance↗

Density-dependent sexual selection in external fertilizers: variances in male and female fertilization success along the continuum from sperm limitation to sexual conflict in the sea urchin Strongylocentrotus franciscanus.

Sperm competition and female choice are fundamentally driven by gender differences in investment per offspring and are often manifested as differences in variance in reproductive success: males compete and have high variance; most females are mated and have low variance. In marine organisms that broadcast spawn, however, females may encounter either sperm limitation or sperm competition. I measured the fertilization success of male and female Strongylocentrotus franciscanus over a range of population densities using microsatellite markers. Female fertilization success first increased and then decreased with mate density, limited at low density by sperm limitation and at high density by polyspermy. Mate density affected variance in fertilization success in both males and females. In males, the variance in fertilization success increased with mate density. In females, the pattern was more complex. The variance in female success increased similarly to males with increased mate density but then decreased to low levels at intermediate densities, where almost all eggs were fertilized. As density increased further, the female variances again increased as polyspermy lowered average fertilization success. Male and female variances differed only at intermediate densities. At low densities, both sexes may be under selection to increase fertilization success; at intermediate densities, males may compete; and at high densities, both sexes may be under selection to increase success by increasing (males) or decreasing (females) likelihood of fertilization during sexual conflict. Only within a narrow range of densities do patterns of sexual selection mirror those typically noted in internally fertilizing taxa.

Animals↗

Signals of Natural Selection Across Regions of Low Recombination in Wild Populations of the Purple Sea Urchin, Strongylocentrotus purpuratus.

Structural variants (SVs) are increasingly recognized as important components of genetic architecture. Yet our understanding of the evolutionary forces maintaining SVs in natural populations is limited. Chromosomal inversions in particular can facilitate local adaptation in populations with high gene flow, including many marine species. The purple sea urchin (Strongylocentrotus purpuratus) is a powerful system to study these dynamics due to its high gene flow, lack of population structure, and broad latitudinal range. We analyzed whole genome sequence data from 137 individuals sampled across seven populations to identify regions of low recombination using scans for elevated linkage disequilibrium and genetic differentiation. Such regions may arise from structural variants, including chromosomal inversions. We identified nine regions showing signatures of reduced recombination, including three way genotype clustering, long range linkage, and hanging bridge patterns frequently associated with inversion polymorphisms. The regions were polymorphic within locations and along the species range with three loci showing concordant signatures of balancing and spatially heterogeneous selection based on enrichment of outliers and distinct patterns of allelic age. Additionally, these loci showed enrichment for genes associated with biomineralization and development. Our results provide the first evidence for regions of low recombination in the purple sea urchin genome, several of which display genomic signatures consistent with structural variants such as chromosomal inversions. These findings add to growing evidence that regions of reduced recombination constitute an important component of standing genetic variation in natural populations and may play a key role in adaptation to heterogeneous environments.

Strongylocentrotus purpuratus↗

Roseivirga echinicomitans sp. nov., a novel marine bacterium isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Roseivirga.

A novel strictly aerobic, heterotrophic, pink-pigmented, non-motile, Gram-negative, oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive marine bacterium, designated strain KMM 6058(T), was isolated from the sea urchin Strongylocentrotus intermedius and studied using a polyphasic taxonomic approach. The G+C content of the DNA of the isolate was 41.3 mol%. The predominant fatty acids were i15:1, i15:0, a15:0 and i17:0 3-OH. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 6058(T) formed a monophyletic clade with Roseivirga ehrenbergii, with 99% similarity. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium should be assigned to the genus Roseivirga as Roseivirga echinicomitans sp. nov. The type strain is KMM 6058(T) (=KCTC 12370(T)=LMG 22587(T)).

Animals↗

Echinicola pacifica gen. nov., sp. nov., a novel flexibacterium isolated from the sea urchin Strongylocentrotus intermedius.

The taxonomic position of three novel marine, heterotrophic, pigmented and agarolytic bacteria with gliding motility, isolated from the sea urchin Strongylocentrotus intermedius, was investigated. 16S rRNA gene sequence analysis revealed that strains KMM 6166, KMM 6172T and KMM 6173 are members of the phylum Bacteroidetes; their nearest neighbours were Belliella baltica and Hongiella marincola (similarities of 94.5 and 93.6 %, respectively). The DNA G+C content of the strains was 44-45 mol%. The predominant fatty acids were C15 : 0 iso, C16:1omega5c, C17:1 iso omega9c, C17:0 iso 3-OH and summed feature 3 (C16:1omega7c and/or C15:0 iso 2-OH). The major respiratory quinone was MK-7. Results of molecular experiments supported by phenotypic and chemotaxonomic data enabled the isolates to be classified as representatives of a novel species in a new genus, for which the name Echinicola pacifica gen. nov., sp. nov. is proposed. Echinicola pacifica is the type species of the genus Echinicola, and its type strain is KMM 6172T (=KCTC 12368T=LMG 23350T).

Animals↗

The genome of the sea urchin Strongylocentrotus purpuratus.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Animals↗

Unexpected diversity displayed in cDNAs expressed by the immune cells of the purple sea urchin, Strongylocentrotus purpuratus.

We recently identified a unique family of transcripts, the 185/333 family, that comprise approximately 60% of the mRNAs induced by coelomocytes from the purple sea urchin in response to immunological challenge from lipopolysaccharide. An analysis of 81 full-length cDNAs revealed 67 unique nucleotide sequences encoding 64 different proteins. Diversity of the transcripts was based on 25 sequence blocks, or "elements," which resulted in 22 different element patterns based on their presence or absence. Furthermore, there was a high level of nucleotide variation within elements, including single nucleotide polymorphisms and insertions/deletions, both of which resulted in amino acid sequence variability. The deduced 185/333 proteins contained an NH2-terminal leader, a glycine-rich region with an RGD motif, a histidine-rich region, and a COOH-terminal region. Two 185/333 genes, identified in the partially assembled Strongylocentrotus purpuratus genome, have two exons. The first encoded the leader, and the second encoded the remainder of the predicted protein. Estimates from quantitative PCR indicated that there were approximately 100 alleles in the diploid genome. These results suggested that the purple sea urchin may have mechanisms for generating high levels of diversity in response to immunological challenge that have not been considered previously.

Amino Acid Motifs↗