Dimorphism of Spermatozoa in the Sea Urchin Strongylocentrotus nudus

A phenomenon of dimorphism in spermatozoa has been revealed for the sea urchin Strongylocentrotus nudus. The spermatozoa are different in the configuration of the circular mitochondrion. The bulk of male gametes (73.4%) have a symmetrical mitochondrion, whereas the remainder spermatozoa (26.6%) have an asymmetrical one. No other ultrastructural differences in the structure of spermatozoa have been revealed. The two types of spermatozoa are supposed to represent different kinds of normal gametes capable of fertilization.     

Fractal Organization of Cultivated in vitro Spiculogenous Cells and Larval Spicules of the Sea Urchin Strongylocentrotus nudus

The morphological patterns of the cultivated cells of primary mesenchyme and the spicules of the larval skeleton of the sea urchin Strongylocentrotus nudus were quantified, and the value of their fractal dimensions (D) was determined with ImageJ 1.20s software. It was shown that during cytodifferentiation, the values of D in the fractal (fractional) dimension, which reflects the complex spatial organization of the spiculogenous mesenchyme elements in two-dimensional space, increase to values close to 1.7. The invertible treatment with cytochalasin, which destroys the system of the actin filaments, suppresses the normal control of biomineralization and causes a complex form of spicules, the fractal dimension of which varies within 1.5–1.6. Thus, the determination of the fractal dimension value serves as evidence of the fractional essence of the patterns studied, quantifies the spatially complex organization of cells and their assemblies during morphogenesis, and allows us to estimate the variation in the spicule morphology after cytochalasin treatment.     

Identification and Characterization of piRNA-Like Small RNAs in the Gonad of Sea Urchin (Strongylocentrotus nudus)

Piwi-interacting RNAs (piRNAs) and their partner PIWI proteins play an essential role in fertility, germline stem cell development, as well as the basic control and evolution of animal genomes. However, research was rare with regard to piRNA population in sea urchin, a model animal intensively used for development and genetics studies. Utilizing Solexa sequencing, we present an identification of 13,051 piRNA-like RNAs expressed in male gonad of Strongylocentrotus nudus. Out of 202 tested RNAs, 94 sequences were confirmed to express in female gonad using microarray assay, suggesting that both male and female gonads are piRNA-like RNA-enriched organs. These RNAs with "U" at the 5' end or "A" at position of 10, in size from 26 to 30 nucleotides, were predominantly 28?nt in length and tend to be clustered in small regions in genome, achieving the longest piRNA-like RNA-enriched region about 5.5?kb in scaffold78427. Alignment results showed 11 RNAs were homologous to the known piRNAs. Furthermore, BLASTn searching against sea urchin repeat element database showed these piRNA-like RNAs matched to 101 types of DNA transposons and retrotransposons, of which SPRP1, Harbinger-N2, piggyBac-N10, SINE2-1, and piggyBac-N11 were the most frequent hit elements, suggesting a transposon silencing function of these piRNA-like RNAs.     

The Effect of Estradiol Dipropionate on the Rate of Protein Synthesis in the Testicle of the Sea Urchin Strongylocentrotus nudus

The effect of estradiol dipropionate on the rate of protein synthesis in the testicle of the mature sea urchin Strongylocentrotus nuduswas studied. The injection of this estrogen considerably intensified ³H-leucine incorporation into the gonad. The change in the level of cell synthetic activity is assumed to be associated with a rise in effective incorporation of ³H-leucine into proteins following an increase in its intracellular level, and with an increase in the protein synthesis rate. The participation of sex hormones in the regulation of protein synthesis in the S. nudusgonad is discussed.     

The Time-Course of Hatching Enzyme Secretion in the Sea Urchin Strongylocentrotus purpuratus

By two independent methods, we have determined approximately the time-course of hatching enzyme secretion in the sea urchin Strongylocentrotus purpuratus . A quick-kill method indicates that a significant fraction of the enzyme is secreted between 90% and 97% of the fertilization-hatching interval. A direct assay method indicates that the remainder of the enzyme is secreted on either side of the 90–97%"window". The entire period of secretion spans from 75% to 100% or more of the fertilization-hatching interval. For embryos raised at 15°C this translates to an interval of 4.8 or more hr.     

Development of Serotonergic Neurons in Embryos of the Sea Urchin, Strongylocentrotus purpuratus

The development of the serotonergic component of the nervous system of larvae of S. purpuratus is traced using indirect immunofluorescence with a polyclonal antibody against the neurotransmitter serotonin. Initially one or two neuroblasts can be detected in the thickened epithelium of the animal plate of late gastrulae (56 hr). The number of immunoreactive cells increases to about eight during formation of the pluteus (85–90 hr). Immunoreactive axons appear simultaneously from all neuroblasts present in the 79 hr prism stage larva and form the apical ganglion. It is proposed that this component of the larval nervous system is derived from a small number of ectodermal cells associated with the apical tuft.     

Allozyme Variability of the Sea Urchin Strongylocentrotus droebachiensis in the Area of Volcanic Hydrotherms

Allozyme variability was studied in 11 polymorphic loci in three samples of the sea urchin Strongylocentrotus droebachiensis using the method of gel electrophoresis. One sample was taken from a population of normal environmental conditions (Avacha Bay, Kamchatka Peninsula), the other two were sampled in Kraternaya Bay (Yankicha Island, the Kuriles): the first sample was from a bottom site where the animals were subjected to continuous stress resulting from the products of volcanic hydrothermal vents and the second sample was from a bottom site where that stress effect was periodical. Statistically significant differences in the genotypic allele frequencies and in levels of heterozygosity of some loci were revealed between the Avacha Bay and Kraternaya Bay samples. Moreover, similar differences, but in other loci, were revealed for the Kraternaya Bay samples. The possible causes of these distinctions are discussed. A conclusion on the primary role of natural selection in the origin of the observed distinctions and on the conformity of the observed data to the hypothesis of selective significance of a part of the allozyme polymorphism is put forth.     

Developmental Expression of Echinonectin, an Endogenous Lectin of the Sea Urchin Embryo

Echinonectin (EN) is a galactose-binding lectin present in eggs and embryos of the sea urchin Lytechinus variegatus . Recent studies have suggested that EN is a hyaline layer protein that may function as a substrate adhesion molecule (SAM) during development. We have used monoclonal and affinity-purified polyclonal antibodies that specifically recognize this protein to determine its spatial and temporal expression during embryogenesis. EN is stored in granules or vesicles in the unfertilized egg. After fertilization, these granules are rapidly redistributed to the apical cytoplasm of the zygote. Our results show that at subsequent stages of development the lectin is expressed by cells of all three germ layers, including cells of the developing gut, coelomic pouches, and ectoderm, and by both primary and secondary mesenchyme cells. In contrast to previous observations based solely upon light level immunofluorescent staining, immunoelectron microscopy demonstrates that EN is localized in intracellular, membrane-bounded vesicles. In epithelial cell types these vesicles have a highly polarized distribution and are found in the apical cortical cytoplasm. In mesenchyme cells the distribution of EN-containing vesicles is not obviously polarized. Steady-state levels of EN protein in the embryo remain almost constant from fertilization to the pluteus larva stage, Metabolic labeling studies show that synthesis of EN in L. variegatus begins immediately after fertilization and continues throughout embryogenesis. Monospecific antibodies raised against L. variegatus EN have also been used to determine whether this lectin is expressed in other echinoid species.     

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.     

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.     

Plagiopyla minuta and Euplotes balteatus, Giliates of the Sea Urchin Strongylocentrotus dröbachiensis

Of 165 sea urchins ( Strongylocentrotus dröbachiensis ) examined in the Mt. Desert Island region, 41 harbored the intestinal ciliate Plagiopyla minuta in considerable numbers. Division in this ciliate is clean; i.e. it is not accompanied by the visible elimination of macronuclear material. Long periods (probably several weeks) of vegetative life appear to alternate with brief periods of intense divisional activity in P. minuta. Twenty-five of these same urchins also contained in their digestive tracts a species of Euplotes which was identified as E. balteatus. This hypotrich was also found commonly in the region as a free-living organism. Dividing specimens were found regularly in the urchin, and such specimens showed the usual macronuclear reorganization which occurs in Euplotes and its near relatives. The evidence indicates that E. balteatus is a facultative commensal, which is perhaps now in the process of acquiring the endozoic habit.     

Effect of Fixed Daylengths on the Photoperiodic Regulation of Gametogenesis in the Sea Urchin Strongylocentrotus purpuratus

Summary

The gametogenic response of the sea urchin Strongylocentrotus purpuratus was compared under treatments of different photoperiod regimes of fixed and seasonal changing daylength. Quantification of gametogenic activity in en- zymatically dissagregated ovaries and in histological sections of testes have shown that the sea urchin is sensitive to both fixed and variable daylengths. After one year at fixed short day (8L:16D) or fixed neutral day (12L:12D) the gonads were ripe and in active gametogenesis, the same as were gonads of animals reared at changing photoperiod and sampled during the short-day phase of the cycle. Under fixed long day (16L:8D) the gonads lacked significant amount of gametes and had the cell constitution found in gonads of animals reared at changing photoperiod and sampled during the long-day phase of the cycle. Measurement of a critical daylength related to the autumnal equinox seems to be part of the mechanism in the photoperiodic regulation of vitellogenic oocyte growth and spermatogenesis in these animals.     

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.     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Developmental Expression of D-Galactoside-Binding Lectin in Sea Urchin (Anthocidaris crassispina) Eggs

The spatial and temporal expression of a sea urchin (Anthocidaris crassispina) egg lectin (SUEL) during early embryogenesis was studied using antiserum raised against SUEL. Western blotting analysis revealed the presence of SUEL in all stages so far examined, from unfertilized eggs to gastrula stage embryos. Immunofluorescence and immunoelectron microscopic observation showed that SUEL was stored in small electron-dense granules which migrated to the cortex within 10 min after fertilization. SUEL was localized in the cortical cytoplasm of the blastomere during cleavage stages and subsequently migrated to the outer surface of the embryo, including the invaginated portion of the gastrula. Immunoelectron microscopic study indicated that SUEL was deposited in the hyaline layer at least at the mid gastrula stage. Migration of SUEL to the cortex was significantly reduced by treatment with cytochalasin B, suggesting that actin filaments play an important role in this translocation. Exogenously added SUEL was adsorbed at the surface of unfertilized eggs and hatched embryos, but not to embryos with fertilization membrane. Lactose inhibited this adsorption, suggesting the presence of an endogenous glycoligand(s) specific for SUEL on the surface of unfertilized eggs and in the hyaline layer. We conclude that SUEL is secreted at a certain stage of embryogenesis and specifically adsorbed to the hyaline layer. Temporal changes in extraembryonic matrices caused by SUEL seem to play an important role in developmental morphogenesis.     

Effects of Light and Covering Behavior on PAX6 Expression in the Sea Urchin Strongylocentrotus intermedius

We studied the diel expression pattern of PAX6 (a structural gene that is commonly involved in the eye development and photoreception of eye forming animals) and the effects of light and covering behavior on PAX6 expression in the sea urchin Strongylocentrotus intermedius. We confirmed that aphotic condition significantly reduced covering behavior in S. intermedius. The diel expression pattern of PAX6 was significantly different in S. intermedius under photic and aphotic conditions. The gene expression of PAX6 significantly deceased in covered S. intermedius both under natural light and in darkness. The present finding provides valuable insight into the probable link between covering and PAX6 expression of sea urchins. Further studies are required to investigate the detailed expression network of light detection involved genes in order to fully reveal the molecular mechanism of the light-induced covering behavior of sea urchins.     

Harvest Refugia in Marine Invertebrate Fisheries: Models and Applications to the Red Sea Urchin, Strongylocentrotus franciscanus

Benthic marine invertebrates with planktonic larvae may exhibitAllee effects in reproduction or recruitment. Hydrodynamic considerationsand experimental evidence suggest that species broadcastinggametes suffer greatly reduced fertilization efficiencies asdensities decline. There is also evidence for some species,including the red sea urchin, that post-dispersal recruitmentsuccess declines at low densities of adults, if adults provideshelter from predators or other sources of mortality. Speciesdisplaying strong Allee effects may be susceptible to catastrophicpopulation collapses with slight increases in mortality dueto harvesting or natural causes. A simple two patch logisticmodel suggests that the establishment of a harvest refuge isnecessary to prevent collapses and maintain sustainable catchesat high levels of harvesting effort. A more detailed, age structuredmodel based on the biology of the red sea urchin, Strongylocentrotusfranciscanus produces similar results. Effects of harvestingstrategies are sensitive to Allee effects caused by both fertilizationefficiency and the disruption of adult spine canopies. Theoreticalrequirements for the size and spacing of marine reserves dependsupon the dispersal abilities of the target species. Multiplereserves, spaced more closely than the average larval dispersaldistance, appear to be an effective and conservative strategyfor maintaining healthy populations and sustainable levels ofharvest.