The effects of prolonged food deprivation on the covering behavior of the sea urchins Glyptocidaris crenularis and Strongylocentrotus intermedius

In this study, we investigated effects of 24 weeks of food deprivation on covering behavior of Glyptocidaris crenularis and Strongylocentrotus intermedius. Neither G. crenularis nor S. intermedius significantly reduced covering behavior during both short- and long-term food deprivation. However, G. crenularis and S. intermedius had significantly different behavioral patterns over the 24 weeks. Covering behavior of G. crenularis greatly increased from the first to the second week, with decreasing reaction time and increasing covering ability, while the covering behavior of S. intermedius generally remained unchanged during the experiment of 24 weeks. As prolonged food deprivation does not significantly affect covering behavior of G. crenularis and S. intermedius, the present study highlights the innate nature and biological significance of covering behavior in sea urchins, providing new insights into its behavioral mechanism.     

The Vertical Distribution of Larvae of the Sea Urchin Strongylocentrotus intermedius in Superficial Desalination Conditions

The vertical distribution of larvae of the sea urchin Strongylocentrotus intermedius in conditions of superficial desalination was studied under laboratory conditions. In the earlier stages of development, the larvae accumulated in the water column zone with a salinity below the optimal level. The long-term observations of the larval accumulations revealed the deceleration of, and abnormalities in, the development and death of the larvae. The larvae at the pre-settling stages changed their behavior: when they reached layers of a salinity unfavorable for their survival or development, the larvae did not enter them and instead moved down instead. Obviously, S. intermedius larvae are only capable of actively choosing their position within the water column with the salinity gradient at the later stages of development.     

The effect of salinity on larval survival and development in the sea urchin Echinometra lucunter

Summary

Reductions in salinity can have adverse effects on larval development and larval survival in some invertebrate taxa but not others. Salinity tolerance of larvae may be particularly important in echinoderms because they are both poor ion regulators and stenohaline. I examined the effect of six levels of salinity (15, 18, 21, 24, 27 and 33 PSU) on survival and rate of development of larvae in the subtropical sea urchin Echinometra lucunter. In the short-term, mortality rate was significantly lower in 33 PSU than in all other salinities except 27 PSU, and it was significantly greater in 15 and 18 PSU than in all higher salinities. In the long-term, daily and cumulative mortality were significantly greater in 15 PSU than in most other salinities over 11 days of development (except for cumulative mortality in 18 PSU). They were significantly greater in 18 PSU than in 21 PSU or 33 PSU over a period of 13 days. Furthermore, daily mortality was significantly greater in 18 PSU than in 24 PSU or 27 PSU at 13 d after fertilization. Daily and cumulative mortality were significantly lower in 33 PSU than in 21, 24 or 27 PSU over a period of 17 days. Although in the control (33 PSU) 75% of larvae completed development to the 8-arm stage at 35 d, no larvae developed further than the 4-arm stage in 18, 21, 24 or 27 PSU; in 15 PSU, ~60% of larvae did not develop further than swimming blastulae. Since prolonged exposure to salinities as high as 27 PSU (frequently recorded in the adult habitat) can result in great larval losses, adaptive behaviours that prevent larvae from entering water layers of low salinity will enhance their chance for survival.     

Bacterial community in the intestine of the sea urchin Strongylocentrotus intermedius during digestion of Macrocystis pyrifera

Denaturing gradient gel electrophoresis (DGGE) was used to study the bacterial community changes in the intestine of the sea urchin Strongylocentrotus intermedius during the digestion of Macrocystis pyrifera. The distinct bands in DGGE gels were sequenced, and the results indicated that the bacterial community in the large and small intestine varied at different periods of digestion. Samples from the large intestine included six specific bands belonging to the genus Psychromonas, whereas samples from the small intestine included eight specific bands representing Psychromonas, Shewanella, Saccharophagus degradans, and Nitrosomonas eutropha. The bacterial flora differed at different periods of digestion. The increase in the microbial community species in the large intestine was not obvious compared with that in the small intestinal microbial community. Several microbes involved in degradation of M. pyrifera were found in the intestine of sea urchin.     

Effects of EHF radiation and cytoactive substances on fertilization and early embryonic development of the sea urchin Strongylocentrotus intermedius

This study examines the influence of extremely high frequency (EHF) electromagnetic radiation (42.2 GHz, 100 μW/cm², impulse modulation 1000 Hz) on the gametes and embryos of the sea urchin Strongylocentrotus intermedius. Preliminary radiation treatment reduces the number of fertilized egg cells and the survival rate of embryos at early stages of development up to the pluteus. Methyluracil reduces the degree of expression of biological effects of EHF radiation.     

Sensitivity of Embryos and Larvae of the Sea Urchin Strongylocentrotus intermedius to Effects of Copper Ions

The effect of copper ions in seawater (0.02 mg/l) on the early stages of development of the sea urchin Strongylocentrotus intermedius was studied. Copper exposure from fertilization or the prism stage retarded development and growth and led to abnormalities in the morphology of the embryos and larvae. However, if development to the pluteus stage proceeded in clean seawater, an increased copper concentration did not inhibit the growth of larvae. If sea urchin embryos at fertilization and the prism stage were maintained for 1–2 days in seawater containing 0.02 mg Cu/l and then transferred to clean seawater, the adverse consequences of this exposure remained present after 48 h.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

Effect of noradrenaline,dopamine and adrenolytics on growth and maturation of the sea urchin,Strongylocentrotus nudus Agassiz

Both noradrenaline and dopamine inhibit growth and maturation of oocytes in sea urchins. The in vitro action of these drugs on gonads was accompanied by a decrease in intensity of incorporation of labelled RNA and protein precursors into oocytes in their cytoplasmic growth. The monoaminergic system appears to participate in the regulation of oogenesis in sea urchins besides the peptidergic neurosecretory system.     

Role of syndecan in the elongation of postoral arms in sea urchin larvae

Ac-SYN is the core protein of a cell surface proteoglycan of the sea urchin Anthocidaris crassispina. To examine the functions of Ac-SYN, embryos were cultured in the presence of affinity-purified antibody against Ac-SYN. At the late pluteus stage, severe inhibition of elongation of the postoral arms was seen in treated embryos compared with control embryos. Blastocoeleic microinjection of the antibody did not affect morphogenesis. The relationship between the number of cells in the postoral arms and the length of the postoral rods was investigated in normal embryos. This showed that postoral arm elongation has two phases: the first phase accompanies the increase in cell numbers while the second does not. The syndecan antibody inhibited the increase in cell numbers in the postoral arms. Furthermore, in the treated embryos, cell numbers continued to increase normally until 31 h post fertilization (hpf), while cell division stopped after 31 hpf. These results suggest that Ac-SYN participates in postoral arm formation via cell division in sea urchin embryos.     

The effect of nicotine on sperm-Egg interaction in the sea urchin: Polyspermy and electrical events

We have studied some of the effects of nicotine on sea urchin eggs, spermatozoa, and their interaction using electrical recording techniques and fertilization-rate experiments. Pretreating eggs with nicotine enhances the fertilization rate, whereas this drug has an inhibitory effect on spermatozoa. Pulse-treated eggs or eggs fertilized in the presence of nicotine give rise to attenuated step depolarizations, which may be attributed to a decrease in membrane resistance (Rm) of the egg or, in the latter case, to an alteration to the spermatozoon. Concurrently, with the change in the step depolarization there is a reduction in amplitude of the fertilization potential (FP) suggesting that the cortical reaction is in some way altered. Nicotine has no effect on the Rm of fertilized eggs or oocytes, where there are no cortical granules. We suggest that nicotine alters the cortex of sea urchin eggs–possibly by causing a partial dissolution of cortical granules–which renders the eggs more receptive to spermatozoa. The reductions in amplitude of the step depolarization and the FP are consequences of this alteration.     

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.     

The dopamine effect on sea urchin larvae depends on their age

Activation of the dopamine type-D₂ receptor in late gastrula of sea urchins is known to decrease the growth rate of post-oral arms of larvae, and, as a result, the phenotype of these larvae mimics that of larvae developing in the abundance of food. Our data indicate that the effect of dopamine on sea urchin larvae is stage-dependent. In our experiment, the early four-armed plutei (96 hours post fertilization, hpf) of Strongylocentrotus intermedius had substantially shorter post-oral arms if they developed from the larvae treated with dopamine at the early pluteus stage (48 hpf), when they had already formed the first dopaminergic neurons, as compared to the plutei from the larvae treated with dopamine at the mid to late gastrula stage (24 hpf), when they did not have any neurons yet. The pre-treatment of larvae in 6-hydroxydopamine, a neurotoxic analog of dopamine that specifically disrupts activity of dopaminergic neurons, prevented the development of the short post-oral arms phenotype in larvae. These results confirm the assumption that dopaminergic neurons play an important role in the development of the short post-oral arms phenotype in sea urchin larvae. Another finding of our study is that the dopamine treatment also affects the growth of the body rods and the overall larval body growth. Based on these observations, we suggest researchers to carefully select the developmental stage, pharmacological agents, and incubation time for experimental manipulation of sea urchin larvae phenotypes through dopaminergic nervous system.     

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.     

Ionophore-induced efflux of sodium and potassium ions from sea urchin eggs

The efflux of K⁺ and Na⁺ from sea urchin eggs during Ca²⁺ ionophore A23187-induced parthenogenesis was studied in a K⁺ and Na⁺-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K⁺ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl₂, 50 mM MgCl₂, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K⁺ and Na⁺ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K⁺ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na⁺ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K⁺ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K⁺ efflux observed egg activation.     

Effects of temperature on hatching rate, embryonic development and early larval survival of the edible sea urchin, Tripneustes gratilla

The temperature tolerances of embryonic and early larval development stages of Tripneustes gratilla were investigated from 13-34°C under laboratory conditions. Zygotes showed unequal cleavage at 13°C, whereas cleavage did not occurred at 34°C. Hatching was observed between 16–31°C with maximum hatching rates observed at 22–29°C. The lower and higher temperature limits for embryonic development were approximately 22°C and 29°C, respectively. Outside of this temperature range, embryos showed abnormality at different incubation times. Early larvae of this species have the ability to survive the higher temperature limit for short periods of time. Prism and 2 arm pluteus larvae survived at temperatures between 30 and 33°C, whereas 4 arm pluteus larvae survived at temperatures between 30 and 36°C for 2 h. These results suggest that the larval temperature tolerance capability of T. gratilla is stage dependent. These findings are important for understanding the life history strategy of this sea urchin in the shallow open water environment.     

Sulfhydryl groups are involved in the activation of sea urchin eggs

Unfertilized sea urchin eggs exposed to the sulfhydryl reagents Ag⁺ or N-ethylmaleimide either elevated fertilizationlike membranes, formed surface protrusions, developed a clear cortical layer devoid of organelles, or cytolysed. The relative fraction of each modification varied from batch to batch and was also dose and time dependent. With Ag⁺ and higher doses of N-EMI (10^?3 M), the most common effect was the elevation of a membrane indicating cortical exocytosis, while at lower doses of N-EMI protrusions were predominant. Glutathione (GSH) protected eggs against these reagents also in a dose-dependent manner. Eggs exposed to equimolar amounts of N-EMI and GSH, which otherwise formed membranes, produced protrusions, while increasing GSH tenfold afforded complete protection. We suggest there are two targets for the sulfhydryl reagents–the first, SH groups on proteins that regulate the release of Ca²⁺ from the intracellular sequestering mechanism which subsequently triggers cortical exocytosis; the second, SH groups on the egg surface that may regulate cortical organization.     

MYP基因在中间球海胆及杂交海胆生殖腺不同发育时期的转录表达差异

摘要: 应用实时荧光定量PCR技术对主要卵黄蛋白(Major yolk protein, MYP)基因在中间球海胆和杂交海胆(中间球海胆♀×光棘球海胆♂)的生殖腺不同发育时期的转录表达差异进行了分析。结果表明, MYP基因在海胆雌雄个体生殖腺中转录水平上的表达差异不明显; MYP基因在中间球海胆生殖腺发育的4个时期的表达呈快速下降的趋势, 而在杂交海胆呈缓慢下降的趋势。在分析的生殖腺4个发育期中, 中间球海胆雌雄个体 MYP 基因的表达量(以18S rRNA为参照)分别从44.55%和41.17%下降到9.59%和1.83%; 杂交海胆的雌雄个体分别从37.66%和36.66%下降到19.22%和12.55%。MYP基因的转录表达量差异与杂交后生殖腺产生的变异密切相关。     

Spiculogenesis in the sea urchin embryo: Studies on the SM30 spicule matrix protein

When proteins isolated from spicules of Strongylocentrotus purpuratus embryos were examined by western blot analysis, a major protein of approximately 43 kDa was observed to react with the monoclonal antibody, mAb 1223. Previous studies have established that this antibody recognizes an asparagine-linked, anionic carbohydrate epitope on the cell surface glycoprotein, msp130. This protein has been shown to be specifically associated with the primary mesenchyme cells involved in assembly of the spicule. Moreover, several lines of evidence have implicated the carbohydrate epitope in Ca²⁺ deposition into the growing spicule. The 43 kDa, spicule matrix protein detected with mAb 1223 also reacted with a polyclonal antibody to a known spicule matrix protein, SM30. Further characterization experiments, including deglycosylation using PNGaseF, two-dimensional electrophoresis, and immunoprecipitation, verified that the 43 kDa spicule matrix protein had a pl of approximately 4.0, contained the carbohydrate epitope recognized by monoclonal antibody mAb 1223 and reacted with anti-SM30. Electron microscopy confirmed the presence of proteins within the demineralized spicule that reacted with mAb 1223 and anti-SM30. We conclude that the spicule matrix protein, SM30, is a glycoprotein containing carbohydrate chains similar or identical to those on the primary mesenchyme cell membrane glycoprotein, msp130.     

Characterization of microsatellite loci for the Australian sea urchin Heliocidaris erythrogramma

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

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.