Pseudocentrotus Depressus Research Articles

Extensive introgression among strongylocentrotid sea urchins revealed by phylogenomics.

Gametic isolation is thought to play an important role in the evolution of reproductive isolation in broadcast-spawning marine invertebrates. However, it is unclear whether gametic isolation commonly evolves early in the speciation process or only accumulates after other reproductive barriers are already in place. It is also unknown whether gametic isolation is an effective barrier to introgression following speciation. Here, we used whole-genome sequencing data and multiple complementary phylogenomic approaches to test whether the well-documented gametic incompatibilities among the strongylocentrotid sea urchins have limited introgression. We quantified phylogenetic discordance, inferred reticulate phylogenetic networks, and applied the Δ statistic using gene tree topologies reconstructed from multiple sequence alignments of protein-coding single-copy orthologs. In addition, we conducted ABBA-BABA tests on genome-wide single nucleotide variants and reconstructed a phylogeny of mitochondrial genomes. Our results revealed strong mito-nuclear discordance and considerable nonrandom gene tree discordance that cannot be explained by incomplete lineage sorting alone. Eight of the nine species examined demonstrated a history of introgression with at least one other species or ancestral lineage, indicating that introgression was common during the diversification of the strongylocentrotid urchins. There was strong support for introgression between four extant species pairs (Strongylocentrotus pallidus ⇔ S. droebachiensis, S. intermedius ⇔ S. pallidus, S. purpuratus ⇔ S. fragilis, and Mesocentrotus franciscanus ⇔ Pseudocentrotus depressus) and additional evidence for introgression on internal branches of the phylogeny. Our results suggest that the existing gametic incompatibilities among the strongylocentrotid urchin species have not been a complete barrier to hybridization and introgression following speciation. Their continued divergence in the face of widespread introgression indicates that other reproductive isolating barriers likely exist and may have been more critical in establishing reproductive isolation early in speciation.

Mannose-binding C-type lectins as defense molecules on the body surface of the sea urchin Pseudocentrotus depressus

We found that the extract of the body wall of the sea urchin, Pseudocentrotus depressus, agglutinate Escherichia coli and is inhibited by mannose. A mannose-binding protein of 22 kDa was purified via affinity chromatography using mannose-agarose. Amino acid sequences obtained by Edman degradation and liquid chromatography quadrupole time-of-flight mass spectrometry followed by de novo sequencing suggested that the protein is a C-type lectin. Products of PCR with a degenerate primer pair and of RACE PCR for the cDNA of the 22 kDa protein were sequenced and produced two full-length cDNA sequences encoding C-type lectins. These two lectins, named P. depressus mannose-binding C-type lectin (PdMBCL) 1 and 2 are composed of 187 and 189 amino acid residues, including signal peptides, respectively, and share 86% identity in their mature form. PdMBCLs agglutinated Lactococcus garvieae, a Gram-positive fish pathogen. Reverse transcription PCR showed that both the genes for the PdMBCLs were expressed in the body wall and in other tissues. Furthermore, the lectins were detected from a rinse of the body surface. Taken together, the present study showed that PdMBCLs function as anti-microbial agents on the body surface of P. depressus.

Compositions of extractive components in the testes and ovaries of various sea urchins: comparisons among species, sexes, and maturational status

We investigated the compositions of free amino acids (FAAs), organic acids (OAs), and nucleotides and their related compounds (NAs) in the gonads of edible sea urchins in Japan. In Hemicentrotus pulcherrimus and Pseudocentrotus depressus, glycine, alanine, lysine, and arginine were abundant FAAs in both sexes before gametogenesis. As gametogenesis proceeded, glycine and threonine showed increasing trends while arginine and histidine showed decreasing trends in both species regardless of sex. Alanine and serine in H. pulcherrimus and isoleucine in P. depressus showed increasing trends regardless of sex. Some other FAAs showed different trends between the sexes. Among the OAs and NAs, adenosine monophosphate, inosine monophosphate, and acetic acid were relatively abundant in H. pulcherrimus testes and ovaries before gametogenesis. In Heliocidaris crassispina, Strongylocentrotus intermedius, and Mesocentrotus nudus, glycine was commonly the most abundant FAA followed by alanine and arginine in the testes and ovaries during early gametogenesis. Compared to the other species, testes and ovaries of Tripneustes gratilla contained lower amounts of arginine and higher amounts of serine and proline. The results of this study could contribute to the development of the aquaculture and seafood industry for sea urchins by accounting for the variation in gonad taste among species, sexes, and according to their maturational status.

The complete mitochondrial genome of Heliocidaris crassispina (A. Agassiz, 1864)

In this study, the complete mitochondrial genome of Heliocidaris crassispina was determined on Illumina HiSeq platform. The genome was 15,678 bp in size and contains 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes and 1 control region (96 bp). The composition of A + T in H. crassispina mtDNA was 59.11%. Except ND6 and 5 tRNAs, the others are on the H-strand. The phylogenetic relationship of 11 species of sea urchins was analyzed using the neighbour-joining method by software MEGA 7.0. H. crassispina was most closely related to Pseudocentrotus depressus.

Early developmental responses of three sea urchin species to tralopyril and its two degradation products

Tralopyril (TLP) is a newly emerged antifouling biocide which rapidly degrades in water. The scientific data on its possible adverse effects to biota is very limited, and even more limited is the ecotoxicity data of its degradation products (DPs). In the present study we investigated the toxicity of TLP and its two main DPs on fertilization and embryogenesis of three sea urchin species: Clypeaster japonicus, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. The species sensitivity to chemicals was investigated and compared. Additionally, the stability of TLP in test medium was examined.TLP in test medium degraded into one single degradation product. The degradation was slower at 17 °C (average incubation temperature for winter species H. pulcherrimus) than at 20 °C (incubation temperature for the other two species). Both DPs at 100 μg/L did not appear toxic, however, TLP highly affected larval development of all tested species. Sensitivity was similar for C. japonicus and P. depressus, but higher toxicity was noticed for H. pulcherrimus. The increased TLP toxicity on exposed H. pulcherrimus embryos could be attributed to the higher TLP stability at lower incubation temperature. Results suggest higher vulnerability to TLP for species spawning in colder seasons. Fertilization test appeared to be less sensitive than the embryotoxicity test.

Effects of elevated carbon dioxide on contraction force and proteome composition of sea urchin tube feet

This study examined how contraction force and protein profiles of the tube feet of the sea urchin (Pseudocentrotus depressus) were affected when acclimated to 400 (control), 2000 and 10,000μatm CO2 for 48days. Acclimation to higher CO2 conditions significantly reduced contraction force of the tube feet. Two-dimensional gel electrophoresis showed that eight spots changed in protein volume: six up-regulated and two down-regulated. Using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight mass spectrometry, three up-regulated spots (tubulin beta chain, tropomyosin fragment, and actin N-terminal fragment) and two down-regulated spots (actin C-terminal fragment and myosin light chain) were identified. One possible interpretation of the results is that elevated CO2 weakened contraction of the tube feet muscle through an alteration of proteome composition, mainly associated with post-translational processing/proteolysis of muscle-related proteins.

Sialic acids as link to Japanese scientists.

This manuscript is dedicated to Prof. Tamio Yamakawa and describes my cooperations on sialic acid-related topics with Japanese scientists during the last 40 years. We studied sialic acids and their O-acetylated derivatives in the sea urchin Pseudocentrotus depressus, in Halocynthia species, and in human and bovine milk. In seafood we mainly searched for N-glycolylneuraminic acid. With synthetic substrates it was shown that sialic acid O-acetylation at C-4 hinders the activity of sialidases, with the exception of viral enzymes. The biosynthesis of Neu5Gc was discussed and the distribution of this sialic acid in dogs followed in modern literature and reviewed regarding their migration. An excellent source of sialic acids is edible bird nest substance (Collocalia mucin) which was used for the synthesis of sialylation inhibitors.

염장처리가 성게 알의 이화학 품질 특성과 산화방지 활성에 미치는 영향

Sea urchin roe obtained from Anthocidaris crassispina and Pseudocentrotus depressus was briefly salt-fermented (5%), followed by ethanol treatment (1%) and the physicochemical properties as well as antioxidant activity were investigated. Compared to raw sea urchin roes, the salted one showed a significantly low amount of water (p＜0.001) high salinity (p＜0.05), ash content (p＜0.001) and Na content (p＜0.001). With salt-fermentation, the redness (p＜0.05) and yellowness (p＜0.001) of roe decreased noticeably, indicating the decomposition of endogenous carotenoids. Accordingly, the salted roe showed a lower DPPH radical scavenging activity than its unsalted counterpart. Additionally, it showed a significantly lower metal-chelating activity (p＜0.05) and metal chelator content (e.g. ortho-phenolics) displayed by a negligible difference in titratable acidity. The salted roe showed significantly increased hardness (p＜0.05) and total reducing capacity (p＜0.001), which were attributed to the protein coagulation and the release of antioxidants bound to macromolecules after the ethanol treatment, respectively.

Effects of the dynein inhibitor ciliobrevin on the flagellar motility of sea urchin spermatozoa.

Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 μM of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin.

Accumulation of the Major Yolk Protein and Zinc in the Agametogenic Sea Urchin Gonad

Sea urchins of both sexes store the nutrients necessary for gametogenesis in nutritive phagocytes of the agametogenic gonad. A zinc-binding protein termed the major yolk protein (MYP) is stored here as two isoforms: the egg-type (predominant in egg yolk granules) and the coelomic fluid-type (a precursor with greater zinc-binding capacity). MYP is used during gametogenesis as material for synthesizing gametic proteins and other components. We investigated its accumulation and relationship to zinc contents in gonads during the non-reproductive season in Pseudocentrotus depressus. MYP constituted most of the protein in coelomic fluid and gonads. Both ovaries and testes grew gradually, accumulating MYP and zinc during the year. Total zinc contents and the ratio of coelomic fluid-type to egg-type protein were higher in ovaries than in testes as gametogenesis approached. Most of the zinc in the coelomic fluid was bound to MYP, and the concentrations of MYP and zinc were elevated toward the onset of oogenesis in the female coelomic fluid. Thus, MYP accumulates in the agametogenic ovaries and testes during the non-reproductive season, playing a role as a carrier to transport zinc to the gonad. Transportation of zinc by MYP is more active in females than in males.

Mammalian Sperm Acrosome Reaction: Where Does It Begin Before Fertilization?

Mammalian Sperm Acrosome Reaction: Where Does It Begin Before Fertilization?

Mass Mortality of Pseudocentrotus depressus Caused by a Bacterial Infection in Summer

Mass Mortality of Pseudocentrotus depressus Caused by a Bacterial Infection in Summer

Nutritional Evaluation of Brown Algae Arame Eisenia bicyclis Sampled at Different Seasons and Places as a Feed for Red Sea Urchin Pseudocentrotus depressus

Nutritional Evaluation of Brown Algae Arame Eisenia bicyclis Sampled at Different Seasons and Places as a Feed for Red Sea Urchin Pseudocentrotus depressus

Notes on the culture of the larvae in the sea urchin, Pseudocentrotus depressus

Notes on the culture of the larvae in the sea urchin, Pseudocentrotus depressus

Examination of Substitute Feed for Chaetoceros sp. at the Larval Stage of Sea Urchin, Pseudocentrotus depressus

Examination of Substitute Feed for Chaetoceros sp. at the Larval Stage of Sea Urchin, Pseudocentrotus depressus

Local Pecurialities as Living for the Red Sea Urchin, Pseudocentrotus depressus of Bishamon Waters off Southern Miura Peninsula, Kanagawa Prefecture, Japan

Local Pecurialities as Living for the Red Sea Urchin, Pseudocentrotus depressus of Bishamon Waters off Southern Miura Peninsula, Kanagawa Prefecture, Japan

The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real-time reverse-transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad.

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.

Zinc‐binding property of the major yolk protein in the sea urchin − implications of its role as a zinc transporter for gametogenesis

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 μM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.