Development of expressed sequence tag-derived microsatellite markers for the sea urchin Hemicentrotus pulcherrimus

The first set of 15 expressed sequence tag-simple sequence repeat markers was developed in sea urchin Hemicentrotus pulcherrimus. Number of alleles per locus ranged from two to 17. The observed and expected heterozygosities ranged from 0.022 to 0.911 and from 0.022 to 0.916, respectively. These informative marker loci will be useful for the assessment of genetic variation and population structure of this species.     

青岛近岸海域马粪海胆摄食的实验生态学研究

对采自青岛近岸海域的马粪海胆从其对各种饵料的选择和摄食吸收、温度与自身湿重对其摄食率的影响以及摄食活动特征等方面进行实验生态学研究。结果表明 ,马粪海胆在多种海藻并存的情况下 ,对海带及裙带菜等褐藻具明显的选择性 ,对石花菜及孔石莼等藻类的喜好性较差 ,但在食物匮乏条件下 ,马粪海胆的食谱则变得相当广泛。马粪海胆对裙带菜及海带的摄食率均明显高于孔石莼 ,但其对孔石莼的饵料吸收率则高于海带及裙带菜。马粪海胆的摄食受温度条件及自身湿重的影响 ,温度偏离适宜范围对摄食有抑制作用 ,日摄食率与湿重呈显著的指数下降关系 ,摄食率与湿重、温度因子可建立具较高可靠性的指数回归模型。马粪海胆对食物的口面吸附与对其摄食密切相关 ,其摄食活动与湿重呈显著的指数下降关系 ,而与排便活动呈显著的正相关 ,随湿重增加排便活动虽呈下降趋势 ,但未达显著水平。自然光照条件下马粪海胆的夜间摄食强度通常高于日间 ,但在全遮盖的无光照条件下这种昼夜摄食差异则变得不显著 ;对摄食活动的连续观测表明马粪海胆的摄食活动受光强变化的影响 ,其摄食活动的高峰出现在光强减弱的早间及傍晚 ,而日间的强光照环境会抑制其摄食活动     

马粪海胆对环境变化的耐受性与选择性研究

对采自青岛近岸的马粪海胆进行温度、盐度、光强与底质等因子的耐受及选择实验。结果表明,青岛近岸马粪海胆的适温范围约在8-22℃,对温度的选择受驯养水温的影响;属窄盐性种类,适盐范围约在30-35;喜好弱光环境,饥饿状态的选择光强（8-25lx）较非饥饿状态（10-35lx）低;对粗沙砾底质具明显的正选择,而对细沙性底质呈明显的负选择。     

Brachyury homolog (HpTa) is involved in the formation of archenteron and secondary mesenchyme cell differentiation in the sea urchin embryo

Sea urchin Brachyury homolog (HpTa) is expressed exclusively in the vegetal plate and secondary mesenchyme cells in the embryos of sea urchin Hemicentrotus pulcherrimus. In order to gain insights into the role of HpTa during sea urchin development, we designed experiments to perturb the embryo by inducing ectopic overexpression of HpTa by injecting fertilized eggs with HpTa mRNA. The overexpression of HpTa resulted in suppression of the formation of vegetal plate and secondary mesenchyme cells. We assume that the interaction of HpTa with unknown factors is required for the activation of the HpTa target genes, and that the excess amount of HpTa proteins produced from injected HpTa mRNA depletes the co-factors. In consequence, the target genes of HpTa would be repressed by the overexpression of HpTa. We suggest that HpTa is involved in the formation of the vegetal plate and the differentiation of secondary mesenchyme cells.     

A laboratory study of the correlation between texture and the speed of locomotion by the sea urchin Hemicentrotus pulcherrimus

This laboratory study examined the relationship between substrate texture and movement speed of the sea urchin Hemicentrotus pulcherrimus. We assessed the movement speed of 14 sea urchins placed on either acrylic, or three types of waterproof sandpaper, on the bottom of a water tank. Images were taken at regular intervals and analyzed to determine the speed of each sea urchin. Light intensity was stronger at one end of the tank. Our analysis showed sea urchins moved away from light, at a speed that was negatively correlated with the roughness of the substrate, with slower movement on rougher surfaces. This result has implications for the design of equipment for capturing sea urchins in areas where their explosive population growth presents a threat to algal growth and reef environments.     

Centriole behavior during meiosis in oocytes of the sea urchin Hemicentrotus pulcherrimus

Ultrastructural changes in the maturing oocyte of the sea urchin Hemicentrotus pulcherrimus were observed, with special reference to the behavior of centrioles and chromosomes, using oocytes that had spontaneously started the maturation division process in vitro after dissection from ovaries. The proportion of oocytes entering the maturation process differed from batch to batch. In those eggs that accomplished the maturation division, it took ~4.5-5 h from the beginning of germinal vesicle breakdown to the formation of a second polar body. Serial sections revealed that a young oocyte before germinal vesicle breakdown had a pair of centrioles with procentrioles, located between the presumed animal pole and the germinal vesicle and accompanied by amorphous aggregates of moderately dense material and dense granules (granular aggregate). Just before germinal vesicle breakdown, a pair of fully grown centrioles located in the granular aggregate, which is present until this stage and then disappears, had already separated from another pair of centrioles. In meiosis I, each division pole had two centrioles, whereas in meiosis II each had only one. The two centrioles in the secondary oocyte separated into single units and formed the mitotic figure of meiosis II. The first polar body had two centrioles and the second had only one. The two centrioles in the first polar body did not form the mitotic figure nor did they separate at the time of meiosis II. These results indicate that, in sea urchins, duplication of the centrioles does not occur during the two successive meiotic divisions and the egg inherits only one centriole from the primary oocyte, confirming the results previously reported for starfish oocytes.     

Vitelline layer lytic activity in sperm extracts of sea urchin,Hemicentrotus pulcherrimus

A factor which dissolves the vitelline layer was extracted from sperm of the sea urchin, Hemicentrotus pulcherrimus. Turbidity of the suspension was reduced when isolated vitelline layers were mixed with this sperm factor. When the mixture was subjected to SDS polyacrylamide gel electrophoresis, some of the protein bands of the vitelline layer were seen to be missing. The lytic activity of the factor was heat labile, completely inhibited by L-1-tosyl-amide-2-phenyl-ethylchloromethyl ketone and partially inhibited by soybean trypsin inhibitor. Chymotrypsin activity was detected, but not trypsin, arylsulfatase, or glycosidase. These results suggest that a chymotrypsin-like enzyme participates in lysis of the vitelline layer by the fertilizing spermatozoon.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.     

硇洲岛海胆可培养细菌的多样性

摘要：【目的】研究南海硇洲岛马粪海胆（Hemicentrotus pulcherrimus）可培养细菌多样性。【方法】采用纯培养法和基于16S rRNA基因序列的系统发育分析对样品中细菌（含放线菌）多样性进行研究。【结果】用补充0～2.0 mol/L NaCl的MA、ISP 2、NA、SWA和HAA培养基从海胆样品中分离到106株细菌菌株,根据形态观察和部分生理生化实验结果去冗余,选取34个代表性菌株进行基于16S rRNA基因序列的系统发育多样性分析。结果表明,这些分离菌株代表21个物种,属于3个大的系     

Establishment of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

In an attempt to estimate the number of pigment precursor cells in sea urchin embryos, DNA synthesis and cell divisions were blocked with aphidicolin from various stages of development. Interestingly, pigment cells differentiated on a normal time schedule, even if the embryos were treated from late cleavage stages on. In most of the embryos treated from 10 h on, 10-15 pigment cells differentiated. Thereafter, the number of pigment cells in the aphidicolin-treated embryos further increased, as the initiation of the treatment was delayed. On the other hand, total cell volumes in the pigment lineage, calculated from the averaged number and diameter of differentiated pigment cells, were almost the same irrespective of the time of the initiation of aphidicolin treatment. This indicated that the increase in the number was caused by divisions of the pre-existing cells in the pigment lineage. Thus, the founder cells that exclusively produce pigment cells could be identified. They are nine times-cleaved blastomeres and specified by 10 h post-fertilization. The obtained results also clarified the division schedule in the pigment lineage; the founder cells divide once (10th) until hatching, and divide once more (11th) by the end of gastrulation.     

Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus)

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.     

Larval arm resorption proceeds concomitantly with programmed cell death during metamorphosis of the sea urchin Hemicentrotus pulcherrimus

Sea urchins are excellent models to elucidate metamorphic phenomena of echinoderms. However, little attention has been paid to the way that their organ resorption is accomplished by programmed cell death (PCD) and related cellular processes. We have used cytohistochemistry and transmission electron microscopy to study arm resorption in competent larvae of metamorphosing sea urchins, Hemicentrotus pulcherrimus, induced to metamorphose by L-glutamine treatment. The results show that: (1) columnar epithelial cells, which are constituents of the ciliary band, undergo PCD in an overlapping fashion with apoptosis and autophagic cell death; (2) squamous epithelial cells, which are distributed between the two arrays of the ciliary band, display a type of PCD distinct from that of columnar epithelial cells, i.e., a cytoplasmic type of non-lysosomal vacuolated cell death; (3) epithelial integrity is preserved even when PCD occurs in constituent cells of the epithelium; (4) secondary mesenchyme cells, probably blastocoelar cells, contribute to the elimination of dying epithelial cells; (5) nerve cells have a delayed initiation of PCD. Taken together, our data indicate that arm resorption in sea urchins proceeds concomitantly with various types of PCD followed by heterophagic elimination, but that epithelial organization is preserved during metamorphosis.This investigation was supported in part by a Keio University special grant-in-aid for innovative collaborative research projects.     

The egg-jelly macromolecule,a fucose sulphate glycoconjugate,originates from the accessory cells of the ovary in the sea urchin Hemicentrotus pulcherrimus

Summary The immunocytochemical localization of the egg-jelly macromolecule, a fucose sulphate glycoconjugate (FSG) that induces the acrosome reaction in spermatozoa, was investigated in ovaries of the sea urchin Hemicentrotus pulcherrimus by use of a polyclonal antibody. The polyclonal antibody reacted with the accessory cells and oocytes in the ovarian lumen. In the accessory cells, evidence of an intense immunohistochemical reaction was observed in many globules of variable density. Products of the specific immunohistochemical reaction were frequently observed in the surface region of oocytes, at a distance from the ovarian wall. At the ultrastructural level, the polyclonal antibody was found to react with the material present in the vacuole-like structures of the globules in the accessory cells. Many gold particles, demonstrating specific immunolabelling, were associated with well-developed microvilli on the vitellogenic oocytes. In the mature oocytes, intense labelling was observed in the jelly coat but not in the vitelline coat. By contrast, oogonia and early oocytes were barely labelled. Quantitative data indicated that the extent of immunolabellings in the surface region of oocytes was very high in the vitellogenic and mature oocytes. In all cases, neither the oocyte cytoplasm nor the subcellular organelles were labelled. These results suggest that FSG is produced by the accessory cells and is deposited initially on the surface of vitellogenic oocytes for the formation of jelly. These findings may provide a new insight into the role of the accessory cells in the reproductive process of the sea urchin. Offprint requests to: N. Suzuki     

Specification and differentiation processes of secondary mesenchyme-derived cells in embryos of the sea urchin Hemicentrotus pulcherrimus

Four types of mesoderm cells (pigment cells, blastocoelar cells, coelomic pouch cells and circumesophageal muscle cells) are derived from secondary mesenchyme cells (SMC) in sea urchin embryos. To gain information on the specification and differentiation processes of SMC-derived cells, we studied the exact number and division cycles of each type of cell in Hemicentrotus pulcherrimus. Numbers of blastocoelar cells, coelomic pouch cells and circumesophageal muscle fibers were 18.0 +/- 2.0 (36 h post-fertilization (h.p.f.)), 23.0 +/- 2.5 (36 h.p.f.) and 9.5 +/- 1.3 (60 h.p.f.), respectively, whereas the number of pigment cells ranged from 40 to 60. From the diameters of blastocoelar cells and coelomic pouch cells, the numbers of division cycles were elucidated; these two types of cells had undertaken 11 rounds of cell division by the prism stage, somewhat earlier than pigment cells. To determine the relationship among the four types of cells, we tried to alter the number of pigment cells with chemical treatment and found that CH3COONa increased pigment cells without affecting embryo morphology. Interestingly, the number of blastocoelar cells became smaller in CH3COONa-treated embryos. In contrast, blastocoelar cells were markedly increased with NiCl2 treatment, whereas the number of pigment cells was markedly decreased. The number of coelomic pouch cells and circumesophageal muscle fibers was not affected with these treatments, indicating that coelomic pouch and muscle cells are specified independently of, or at much later stages, than pigment and blastocoelar cells.     

Seasonal variations in the production of the egg-jelly macromolecule,a fucose sulphate glycoconjugate,by the accessory cells in the ovary of the sea urchin Hemicentrotus pulcherrimus

In the sea urchin Hemicentrotus pulcherrimus, the egg-jelly macromolecule, a fucose sulphate glycoconjugate (FSG) that induces the acrosome reaction in spermatozoa, originates from the accessory cells in the ovary. In the present study we examined the seasonal variations in the distribution of FSG in the ovary by immunocytochemistry with a polyclonal antibody. An enzyme-linked immunosorbent assay indicated that FSG was present in supernatants of extracts of ovaries throughout the development of the ovary. However, the immunohistochemical study showed that there are marked seasonal changes in the distribution of FSG in ovaries. The polyclonal antibody reacted strongly with globules of accessory cells before the beginning of the breeding season (August to December). During the breeding season (February to April), the immunohistochemical reaction was found on the surface of oocytes but was weak in the accessory cells. At the ultrastructural level, the antibody reacted with globules of variable density in accessory cells. Intense immunolabelling was observed in the vacuole-like structures of the globules. Sometimes, products of the specific immunocytochemical reaction were found in the Golgi apparatus in these globules. Quantitative examination indicated that FSG was actively produced by the accessory cells from the late non-breeding season to the pre-breeding season. These results suggest that there are marked seasonal variations in the production of FSG by the accessory cells in the sea urchin ovary. These findings also provide new evidence that accessory cells exhibit dynamic changes during the reproductive process in the sea urchin.     

Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos

• 1.1. Arylsulfatase was extracted from sea urchin (Hemicentrotus pulcherrimus) plutei and purified to electrophoretical homogeneity by means of DEAE-cellulose, acetone fractionation and Sepharose CL-6B, successively.
• 2.2. The molecular weight of this enzyme was approx, 670,000. The molecular weight of a single subunit was approx. 63,000. The K_m value for p-nitrophenyl sulfate was 0.59 mM.
• 3.3. This enzyme was competitively inhibited by the sulfate ion and was classified as the type II arylsulfatase. The pH optimum was between 5.0 and 6.0.