Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus)

Summary Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.     

Predation of the sea urchin Heliocidaris erythrogramma by rock lobsters (Jasus edwardsii) in no-take marine reserves

The formation of sea urchin ‘barrens’ on shallow temperate rocky reefs is well documented. However there has been much conjecture about the underlying mechanisms leading to sea urchin barrens, and relatively little experimentation to test these ideas critically. We conducted a series of manipulative experiments to determine whether predation mortality is an important mechanism structuring populations of the sea urchin Heliocidaris erythrogramma in Tasmania. Tethered juvenile and adult sea urchins experienced much higher rates of mortality inside no-take marine reserves where sea urchin predators were abundant compared to adjacent fished areas where predators were fewer. Mortality of tagged (but not tethered) sea urchins was also notably higher in marine reserves than in adjacent areas open to fishing. When a range of sizes of sea urchins was exposed to three sizes of rock lobsters in a caging experiment, juvenile sea urchins were eaten more frequently than larger sea urchins by all sizes of rock lobster, but only the largest rock lobsters (> 120 mm CL) were able to consume large adult sea urchins. Tagging (but not tethering) juvenile and adult sea urchins in two separate marine reserves indicated that adult sea urchins experience higher predation mortality than juveniles, probably because juveniles can shelter in cryptic microhabitat more effectively. In a field experiment in which exposure of sea urchins to rock lobster (Jasus edwardsii) and demersal reef fish predators was manipulated, rock lobsters were shown to be more important than fish as predators of adult sea urchins in a marine reserve. We conclude that predators, and particularly rock lobsters, exert significant predation mortality on H. erythrogramma in Tasmanian marine reserves, and that adult sea urchins are more vulnerable than smaller cryptic individuals. Fishing of rock lobsters is likely to reduce an important component of mortality in H. erythrogramma populations.     

Phagocytic amoebocyte sub populations in the perivisceral coelom of the sea urchin Lytechinus variegatus (Lamarck, 1816)

The echinoderms are deuterostomic animals with a nonspecific immune system similar to that of vertebrates. Among coelomocytes, phagocytic amoebocytes have a key role in the nonspecific immune response in sea urchin, being responsible for microorganisms elimination through phagocytosis and also for humoral secretions of a wide spectrum. Sub-populations of phagocytic amoebocytes (PA) have been previously described and two distinct sub populations in the oral (OR) and aboral (AB) regions of the perivisceral coelom of L.variegatus in the present study were found. In the OR there is a higher number of PA with higher phagocytic capacity after 30 minutes of incubation with yeast and higher percentage of intranuclear iron crystalloids. The germicide capacity under the fluorescence technique did not show any difference. SDS-PAGE analysis showed different protein patterns between coelomocytes of OR and AB. Gravitational force had no effect in PA distribution and no physical barrier was found in the perivisceral coelom. The other coelomocyte (vibratile cells, red spherulocytes and white spherulocytes) populations were not different in OR compared with AB in their distribution. Some aspects of the possible causes of the differences found for PA are discussed in the paper.     

Larvae and postlarvae production of the green-white sea urchin Lytechinus variegatus (Echinodermata: Echinoidea) in culture conditions

We evaluated the biological feasibility of massive larvae and postlarvae production of the Caribbean green-white urchin Lytechinus variegatus. Experiments were designed to choose the initial larval density and microalgae diets under culture, to study metamorphosis, postlarval and juvenile growth. Massive production of competent larvae 650 microm long at 12-13 days is possible using larval densities of 0.25 to 1 larva/ml. The microalgae Rhodomonas sp. was suitable for the optimization of larval growth and survival. Metamorphosis of 100% of the larvae can be induced with films of bentic diatoms (Navicula sp. and Amphora sp.), after 96 h; however, diatoms are not adequate for postlarval development and a food supply of Ulva lactuta is necessary for proper growth. For juveniles, a diet of macroalgae (U. lactuta) and/or commercial marine shrimp culture pellet food is enough for growth, but the best results were obtained with shrimp or U. lactuta used alone (85-86%, against 46% with the mixed diet). We recommend future experiments on nutritional requirements to optimize growth of these and subsequent stages.     

Population of Lytechinus variegatus (Echinoidea: Toxopneustidae) and structural characteristics of seagrass of Thalassia testudinum in Mochima Bay,Venezuela)

To compare the general features of Thalassia testudinum seagrass at Mochima Bay with sea urchin (Lxtechinus variegatus) abundance and distribution, three T. testudinum seagrass beds were selected, from the mouth (strong wave exposure) to the inner bay (calm waters). Each site was surveyed by using 5 line transects (20 m long) parallel to the coast and 1 m2 quadrats. In situ measurements of T. testudinum cover, shoot and leaf density were taken. Estimation of dry biomass for each seagrass fraction (leaves, rhizomes and roots) and leaf length were obtained from 25 vegetation samples extracted per site using cores (15 cm diameter). A multivariate analysis of variance (Manova) and a less significative difference test (LSD) were performed to examine differences between sites and within sites at different depths. A stepwise multiple regression analysis was done, dependent variable was sea urchin density; independent variables: vegetation values at each site. The only seagrass species found in the three sites was T. testudinum, and cover was 56-100%, leaf density 100-1000 leaf/m2, lengths 6-18.8 cm and shoot density 20-475 shoots/m2. The highest sea urchin densities were found at Isla Redonda and Ensenada Toporo (1-3.6 ind/m2), the lowest at Playa Colorada (0.6-0.8 ind/m2). Significant differences in seagrass features between sites were obtained (Manova p < 0.001), but not between depths (Manova p < 0.320). The regression coefficient between sea urchin density and seagrass parameters was statistically significant (r2 = 0.154, p < 0.007), however, total biomass was the only variable with a significant effect on sea urchin distribution (beta = 0.308, p < 0.032). The other variables did not explain satisfactorily L. variegatus abundance and distribution.     

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.     

Nucleotide sequence of a 5S ribosomal RNA gene in the sea urchin Lytechinus variegatus.

The nucleotide sequence of a cloned DNA fragment corresponding to a gene for 5S ribosomal RNA from the sea urchin Lytechinus variegatus has been determined. This sequence is representative of the dominant species of 5S rRNA labelled in vivo with 32pO4 during the cleavage stage of Lytechninus embryonic development.     

Diameter-weight relationship and chromatic proportion of the sea urchin Lytechinus variegatus (Echinoidea: Toxopneustidae) in the islands of Margarita and Cubagua, Venezuela

In Margarita and Cabagua Islands, Venezuela, the sea urchin Lytechinus variegatus is consumed as food but few studies concern its biology. Between January 1999 and September 2000, 25 localities of Margarita and Cubagua islands we visited to determinate the relationship between diameter and weight of living urchins according to their coloration green or white. A total of 5 066 urchins (3 421 captured in Margarita and 1 645 in Cubagua) were measured. There is no significant difference in the weight-diameter relations of green or white sea urchins of both islands. The weight-diameter equation is P = 0.00246 D252. Utilizing a quadrant (0.5 x 0.5 m) monthly samples of urchins living in a 2 m2 of surface were taken to estimate the abundance of the specimens according their color (N = 6 948 urchins). The green urchins were more abundant than the white urchins, in a 3:2 proportion.     

Effects of inorganic and organic phosphate exposure on aspects of reproduction in the common sea urchin Lytechinus variegatus (Echinodermata: Echinoidea)

The common nearshore sea urchin Lytechinus variegatus is capable of surviving exposure to inorganic phosphate concentrations as high as 3.2 mg/l(-1) and organic phosphate concentrations of 1,000 mg/l(-1). However, chronic exposure to low, medium, or high sublethal concentrations of these phosphates inhibits gonadal tissue indices and spawning activity while altering biochemical composition of gonads, reducing size frequencies of oocyte diameters, and changing gonadal volume fractions. Gonad indices declined significantly in individuals maintained in all phosphate concentrations after both one- and two-month exposures, while percentages of ripe individuals (oozing gametes upon dissection) were reduced after a two-month exposure in individuals maintained in medium and high organic phosphate concentrations. Levels of carbohydrates and lipids were lower in gonads of individuals maintained in all concentrations of both phosphates. Size frequency distributions of oocyte diameters revealed a dramatic decrease in oocyte size with increasing concentrations of both phosphates. Gonadal volume fractions of developing male and female gametes decreased with exposure to increasing phosphate levels. Volume fractions of nutritive phagocytes declined in testes of individuals held in the highest concentration of organic phosphate but displayed no significant change in ovaries. Volume fractions of mature gametes also decreased in gonads of individuals exposed to increasing concentrations of inorganic phosphate, but they remained constant in individuals exposed to all concentrations of organic phosphate. These findings indicate that shallow-water populations of L. variegatus subjected to inorganic and organic phosphate pollutants will exhibit stress that may impair reproductive output, gametogenesis, and spawning in the natural environment.     

The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea)

The embryos of the sea urchin Lytechinus variegatus are capable of surviving chronic exposure to inorganic sodium phosphate and organic triethyl phosphate concentrations as high as 6 and 1000 mg l(-1) seawater, respectively. However, chronic exposure to sub-lethal concentrations of these phosphates may cause arrested or abnormal embryonic development. We measured fertilization success and percentages of normal, arrested and abnormal embryos exposed to low, medium and high sub-lethal concentrations of inorganic and organic phosphate. Fertilization success was significantly reduced in all phosphate treatments. After attaining the 4-cell stage, embryos exposed to the highest phosphate concentrations displayed arrested development. Percentages of abnormally developing embryos showed a strong concentration dose-response with a significant increase in abnormal embryonic development with increasing phosphate concentration. Overall, these results indicate that the gametes and embryos of L. variegatus may provide a rapid and sensitive model bioassay for the evaluation of phosphate pollutants in marine systems. Our findings also indicate that shallow-water populations of L. variegatus spawning in areas subjected to inorganic and organic phosphate pollutants may suffer detrimental effects on fertilization and embryonic development.     

The proteome of the developing tooth of the sea urchin, Lytechinus variegatus: mortalin is a constituent of the developing cell syncytium

Echinoderm teeth are continuously growing calcite-mineralized tissues of complex structure. Two features are of special interest: (1) cell division takes place in a restricted aboral domain, the plumula, and the cells immediately merge into multinucleated syncytial layers; (2) the major part of the heavily mineralized tooth elongates and moves towards the adoral incisal tip continuously as the syncytial cells actively expand the syncytium and intermembrane mineral phase. As the first step to understanding the nature of the mineralization processes, we have isolated the proteins of the plumula and of the mature mineralized portions of the tooth, and begun their characterization. Peptide sequences were used to screen a plumula cDNA library by polymerase chain reaction. One primer set yielded a prominent amplified product which was cloned, and sequenced. Comparison with the nucleotide and protein data banks revealed the protein to be Mortalin, a member of the hsp-70 family, with >75% of its sequences identical to that of human mortalin. Immunocytochemical localization of mortalin within the plumula, using Anti-human Grp75, showed staining of the odontoblast cytosol and matrix at the point where syncytial formation was occurring. The cytosol of the syncytial layers was weakly stained. The nuclei within the syncytia were stained at their periphery. In the mature part of the tooth, the perinuclear staining of the nuclei was more prominent. We conclude that mortalin is involved in syncytium formation and maintenance. The urchin mortalin has a distinctive aspartic acid and serine-rich C-terminal domain that may link it to the mineralization process.     

Population structure,recruitment, and mortality of the freshwater crab Dilocarcinus pagei Stimpson, 1861 (Brachyura,Trichodactylidae) in Southeastern Brazil

Freshwater crabs of the family Trichodactylidae are widely distributed in major river basins of the South American continent. The population structure of one species, Dilocarcinus pagei, was analyzed in an artificial reservoir in Southeastern Brazil, recording data on the sex ratio, recruitment, and mortality. A total of 1339 crabs were collected and included 804 males and 535 females (3 ovigerous and 4 with hatchlings); the sex ratio was 1:0.61 (males: females). Two modes of male and female carapace width (CW) were recorded, with means of 14.4 mm (n = 407) and 38.9 mm (n = 394) for males and 17.9 mm CW (n = 269) and 39.2 mm (n = 267) for females. The mean size of males (CW = 26.6 ± 13.8 mm) was significantly larger than that of females (CW = 28.5 ± 12.8 mm). The data indicated that recruitment occurs in summer (January–March), with the reproductive period in spring (October–December), periods with the highest rainfall and temperature values in the region. Reproduction leads to the death of the parents, influencing the sex ratio, which oscillates mainly during the reproductive period.     

Effects of hypercapnia on aspects of feeding,nutrition, and growth in the edible sea urchin Lytechinus variegatus held in culture

Land-based aquaculture facilities experience occasional hypercapnic conditions due to the accumulation of the metabolic waste product carbon dioxide. Pre-gonadal Lytechinus variegatus (horizontal diameter?=?20?mm) were exposed to control (608?μatm pCO₂, pH 8.1) or hypercapnic conditions (1738?μatm pCO₂, pH 7.7) in synthetic seawater for 14?weeks. Sea urchins exposed to hypercapnic conditions exhibited significantly slower growth (reduced dry matter production), primarily due to reduced test production. Higher fecal production rates and lower ash absorption efficiency (%) in individuals exposed to hypercapnic conditions suggest the ability to process or retain dietary carbonates may have been affected. Significant increases in neutral lipid storage in the gut and increased soluble protein storage in the gonads of individuals exposed to hypercapnic conditions suggest alterations in nutrient metabolism and storage. Furthermore, organic production and energy allocation increased in the lantern of those individuals exposed to hypercapnic conditions. These results suggest chronic exposure to hypercapnic conditions alters nutrient allocation to organ systems and functions, leading to changes in somatic and reproductive production.     

Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.     

Impact of sewage pollution on two species of sea urchins in the Mediterranean Sea (Cortiou,France): Radial asymmetry as a bioindicator of stress

BackgroundThe surroundings of the Cortiou sewage are among the most polluted environments of the French Mediterranean Sea (Marseilles, France). So far, no studies have precisely quantified the impact of pollution on the development of organisms in this area.MethodsWe used a fluctuating asymmetry (FA) measure of developmental instability (DI) to assess environmental stress in two species of radially symmetric sea urchins (Arbacia lixula and Paracentrotus lividus). For six sampling sites (Cortiou, Riou, Maire, East Maire, Mejean, and Niolon), levels of FA were calculated from continuous and discrete skeletal measures of ambulacral length, number of pore pairs and primary tubercles.ResultsFor both species, the most polluted sampling site, Cortiou, displayed the highest level of FA, while the Maire and East Maire sampling sites displayed the lowest levels. A. lixula revealed systematic differences in FA among sampling sites for all characters and P. lividus showed differences in FA for the number of primary tubercles.ConclusionsStatistical analyses of FA show a concordance between the spatial patterns of FA among sampling sites and the spatial distribution of sewage discharge pollutants in the Cortiou area. High developmental stress in these sampling sites is associated with exposure to high concentrations of heavy metals and many harmful organic substances contained in wastewater. FA estimated from structures with complex symmetry appears to be a fast and reliable tool to detect subtle differences in FA. Its use in biomonitoring programs for inferring anthropogenic and natural environmental stress is suggested.     

The effect of feeding frequency on consumption of food,absorption efficiency,and gonad production in the sea urchin Lytechinus variegatus

The frequency of feeding in the field is variable in sea urchins, ranging from nearly continuous to diel or intermittent. It is essential to know the effect of feeding interval on physiological and metabolic processes to understand the basis for production. Lytechinus variegatus (50 mm horizontal diameter) were collected in January 1999 and held in closed-circuit aquaria at 25 degrees C and 35 per thousand salinity. After 9 days without food, individuals were fed one of three treatments: food available ad libitum, food available for 1 day every 2 days or food available for 1 day every 4 days for 28 days. The rate of food consumption per day of all individuals was high the first week of feeding. It then decreased to a lower, constant rate in those fed ad libitum but remained high in those fed one day every 2 or 4 days. The total amount eaten was directly related to frequency of feeding. The apparent dry matter digestibility (absorption efficiency) did not vary with frequency of feeding. As the total amount of energy absorbed was directly related to the frequency of feeding, the increase in the rate of food consumption does not compensate for a decrease in frequency of feeding. Gonad production efficiency was directly related to frequency of feeding. Gonad gross production (assimilation) efficiencies were 8.4, 3.9 and 3.4% for individuals fed every day, or fed one day every 2 and 4 days, respectively. The corresponding gonad net assimilation efficiencies were 12.5, 5.5, and 4.8%. A decrease in frequency of food availability results in use of a greater proportion of the food consumed for maintenance and less for gonad production.     

Resistance of the heart sea urchin Echinocardium cordatum (Echinoidea: Spatangoida) to extreme environmental changes

The ability to survive under extreme environmental conditions was studied in the adults of the heart sea urchin Echinocardium cordatum (Pennant). At seawater temperatures of 13.3 to 14.8°C and salinity of 33.2–33.4‰, being devoid of the possibility to burrow into the sand or eat, some sea urchins died on day 5 and all individuals had perished by the end of day 8. At a temperature of 19°C, the salinity tolerance range of adults was limited to 33–28‰. Only 30 to 20% of sea urchins transferred to a solid substrate survived for 7 days at a salinity of 33 to 24‰, but all of them perished toward the end of day 8.