Polyfluorochrome marking slows growth only during the marking month in the green sea urchin Strongylocentrotus droebachiensis

Abstract. Used singly, the fluorochrome tags tetracycline and calcein have yielded important insights into sea urchin biology, especially regarding growth. We present a new method of tagging using sequential fluorochrome markers, as well as a more precise method of quantifying growth. Such polyfluorochromes enable repeated markings that allow measurement of multiple growth points and unique identification of individuals or groups. We marked sea urchins, Strongylocentrotus droebachiensis , with four fluorochromes: alizarin complexone, calcein, calcein blue, and tetracycline. All fluorochromes marked both by injection and immersion. We examined the clarity of the mark produced with low, metabolically scaled doses, and higher doses similar to those that have been previously used. We tested the effect of fluorochromes on survival, growth, jaw size, and gonad size by marking a size range (3.9–44.3 mm in diameter) of urchins with either one or all four fluorochromes. We quantified growth using a nominal diameter, that is a fitted constant, times the cube root of weight, which increased the precision of measurements by a factor of six relative to measured diameter. Growth rate was a decreasing function of diameter except for a growth lag in the smallest urchins. Growth rate data for all sizes were fitted using: gamma distributions; Tanaka functions; and, for larger sizes, straight lines (von Bertalanffy model). All treatments produced clear marks, with higher doses producing more reliably clear marks. Tetracycline marking did not affect growth; other treatments produced only transient slowing of growth in the marking month. Growth rate, survival, gonad production, and jaw weight did not differ between control and treatments during the following 6 months. Thus, polyfluorochromes produce reliable marks that do not significantly affect growth or gonad production.     

Larval settlement of the green sea urchin, Strongylocentrotus droebachiensis, in the southern Gulf of Maine

Abstract. Settlement timing and differential settlement for the larval stage of the green sea urchin, Strongylocentrotus droebachiensis , in the southern Gulf of Maine was studied during the summer of 1996. Settlement densities on astroturf panels were highest in June and early July (13 to 37 m ⁻² d⁻¹), and peaked in mid-June (199 m⁻² d⁻¹). Settlement was low to nonexistent from mid-July through August (0 to 2 m⁻² d⁻¹). During the peak in settlement, no selection for substrate type was observed. In the remainder of the settlement period, differential settlement occurred, with a preference for substrate covered with live coralline algae. Test diameter of newly settled urchins varied among the substrates, with urchins settling on live coralline algae having the largest test diameter (0.43 ± 0.01 mm). There were no differences in test diameter among the different weeks in which sampling was done. Sustained onshore winds occurred only during peak settlement, suggesting that wind drift currents may concentrate larvae and influence patterns of larval settlement.     

Egg longevity and time-integrated fertilization in a temperate sea urchin (Strongylocentrotus droebachiensis)

Recent field experiments have suggested that fertilization levels in sea urchins (and other broadcast spawners that release their gametes into the water column) may often be far below 100%. However, past experiments have not considered the potentially positive combined effects of an extended period of egg longevity and the release of gametes in viscous fluids (which reduces dilution rates). In a laboratory experiment, we found that eggs of the sea urchin Strongylocentrotus droebachiensis had high viability for 2 to 3 d. Fertilization levels of eggs held in sperm-permeable egg baskets in the field and exposed to sperm slowly diffusing off a spawning male increased significantly with exposure from 15 min to 3 h. In a field survey of time-integrated fertilizations (over 24, 48, and 72 h) during natural sperm release events, eggs held in baskets accrued fertilizations over as much as 48 h and attained fairly high fertilization levels. Our results suggest that an extended period of egg longevity and the release of gametes in viscous fluids may result in higher natural fertilization levels than currently expected from short-term field experiments.     

Respiration of mitochondria from the gonads of the sea urchin, Strongylocentrotus droebachiensis: the effects of petroleum fractions on oxygen consumption

The respiration of mitochondria from gonads of Strongylocentrotus droebachiensis has been studied in the presence of 2-oxoglutarate, glutamate and succinate. The rate of oxygen consumption increased with temperature between 4 and 20 degrees C. Removal of gametes from ripe gonads was necessary to obtain good respiring mitochondria. Equilibration of respiration media with various petroleum fractions reduced the rate of oxygen consumption and the coupling of O2 consumption to ADP utilization. Lighter fractions (leaded gasolines, unleaded gasoline and related fractions) were more deleterious than medium fractions (kerosene and fuel oil). Heavy fraction (bunker C, vacuum bottoms and crude oil) were least deleterious. Tetraethyl lead contributed to the toxicity of the leaded fractions.     

Nutritive phagocyte incubation chambers provide a structural and nutritive microenvironment for germ cells of Strongylocentrotus droebachiensis, the green sea urchin

Here we characterize the germinal epithelia of both sexes of Strongylocentrotus droebachiensis, the green sea urchin, throughout its annual gametogenic cycle, using light and electron microscopy and cytochemistry. In both sexes, germinal epithelia include two interacting cellular populations: nutritive phagocytes (NPs) and germ cells. After spring spawning, NPs accumulate nutrients; amitotic oogonia and often mitotic spermatogonia occur in clusters beneath NPs; and subsequent gametogenic stages are residual or absent. During the summer, NP nutrients are mobilized for use in vitellogenesis by residual primary oocytes or to support limited spermatogenesis. In addition, some residual primary oocytes may degenerate and be phagocytized by NPs. Significant nutrient mobilization from NPs and substantial gonial cell mitoses (indicative of new gametogenesis) occur in the fall. In both sexes, all of these changes are facilitated by NPs that form basal incubation chambers near the gonadal wall and within which germ cells are surrounded by nutrients released from the NPs. In females, germ cells at several stages of gametogenesis may be housed in separate chambers in the same NP. Primary oocytes also carry out jelly coat formation, meiosis, and cortical granule translocation within NP incubation chambers. In males, many NPs cooperate to provide large continuous chambers that contain spermatogenic cells at diverse stages. In both sexes these chambers persist throughout the year and isolate gametogenesis from the gonadal lumen. NPs become slender and shorten as their nutrients are depleted. Ova or spermatozoa are stored in the gonadal lumen. Post-spawning, NPs phagocytize differentiated germ cells while simultaneously enclosing intact gonial and residual gametogenic cells in basal chambers near the gonadal wall. In light of our observations, we suggest investigating proteins that may be important in the structural, phagocytic, and nutritive functions of NPs and for which corresponding genes have already been identified in the genome of S. purpuratus, the closely related purple sea urchin.     

Stereological analysis of nutritive phagocytes and gametogenic cells during the annual reproductive cycle of the green sea urchin, Strongylocentrotus droebachiensis

Abstract. The germinal epithelium of sea urchin gonads contains two interdependent populations of cells: somatic cells called nutritive phagocytes (NPs) and germ cells—ovarian and testicular gonial cells and their derivatives, gametocytes and gametes. Annually, NPs vary their structure and function to produce a changing microenvironment for germ cells during gametogenesis and after spawning. Here, we describe seasonal changes in the NPs as they interact with germ cells during successive gametogenic stages in both sexes of the green sea urchin, Strongylocentrotus droebachiensis. Monthly samples were collected from the Gulf of Maine at a depth of 10 m and analyzed with light microscopy and stereology. Shorter day length and falling seawater temperatures were correlated with nutrient mobilization from NPs, initiation of gonial cell mitosis, and subsequent gametogenesis. During gametogenesis, NPs in both sexes were depleted of nutrients and eventually phagocytize residual ova or spermatozoa. Observations from this study are important for understanding both the cellular aspects of the reproductive biology of sea urchins and those environmental factors that affect the onset and progress of gametogenesis.     

Sperm polymorphism within the sea urchin Strongylocentrotus droebachiensis: divergence between Pacific and Atlantic oceans

The rapid evolution of traits related to fertilization such as sperm morphology may be pivotal in the evolution of reproductive barriers and speciation. The sea urchin Strongylocentrotus droebachiensis has a circumarctic distribution and shows substantial genetic subdivision between northeastern Atlantic populations and northwestern Atlantic and Pacific populations. Using transmission electron microscopy, we show here that sperm shape, size, and ultrastructure differ markedly among populations of S. droebachiensis from different oceans and reflect patterns of genetic divergence. Sperm nuclei from northwestern Atlantic and Pacific populations were longer and narrower than those from the northeastern Atlantic. We additionally demonstrate population-level differences in the amount and location of filamentous actin (F-actin) prior to the occurrence of the acrosome reaction. Sperm from Pacific and northwest Atlantic populations differed from that of all other echinoids examined in that intact sperm contains a partly preformed acrosomal process, a structure more closely resembling the acrosomal rod seen in some molluscs. Immunofluorescent studies using anti-bindin antibodies and the F-actin-specific stain phalloidin confirmed these findings. Divergence of reproductive traits such as sperm morphology may be related to divergence in gamete compatibility and genetic divergence, and could represent the first stages of speciation in free-spawning marine invertebrates.     

Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis)

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+- ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.     

The influence of diet on the oxygen uptake of the sea urchins,Tripneustes ventricosus and Strongylocentrotus droebachiensis

• 1.1. The influence of different natural foods on the rate of oxygen uptake of two species of sea urchins was examined.
• 2.2. The rate of oxygen uptake of specimens of Tripneustes ventricosus was greater in those fed with the brown alga, Saryassum sp., than in those fed with the angiosperm, Thalassia testudinum.
• 3.3. When specimens of Strongylocentrotus droebachiensis were fed following a period of food deprivation, the increase in the rate of oxygen uptake was higher in individuals fed with the brown alga, Nereocystis luetkeana, than in individuals fed with the angiosperm, Zostera marina. This difference is attributed to a higher specific dynamic action resulting from a greater rate of food absorption in urchins fed with N. luetkeana.

     

Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis

Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate.     

A morphological analysis of the first cleavage mitotic cytoskeleton isolated from sea urchin embryos (Strongylocentrotus droebachiensis)

Morphological changes in the mitotic cytoskeleton (MC) that occurred through the course of the first cleavage of Strongylocentrotus droebachiensis, cultured at 8°C or at 0°C, temperatures within the natural range for this species, have been investigated. Electron microscopy of MCs isolated from zygotes has revealed that they consisted largely of microtubules (mts). Thus, the morphology of these MCs is derived from the arrangement of the mts which form them. During anaphase, astral rays elongated while kinetochore fibers shortened. Asters enlarged during anaphase as a result of two events: astral ray lengthening and centrosphere enlargement. At the end of anaphase, asters of 8°C MCs filled the entire cell volume. The pattern of changes that occurred in 8°C mitotic apparatuses (MAs) also occurred in 0°C MCs. The observation of asters in 0°C MCs is contradictory to that of Stephens ('72b), who reported that 0°C MCs in this species were anastral. However, in 0°C metaphase MCs, the astral rays and spindle fibers were not as long as those in 8°C MCs. Also in 0°C MCs, the centrosphere was largely filled with dense material, whereas the centrosphere in 8°C MCs was larger and contained little dense material. Asters of 0°C MCs did not attain a large enough size to fill the egg volume completely, as did asters of 8°C MCs.     

Effects of location,exposure and physical structure on juvenile recruitment of the sea urchin Strongylocentrotus droebachiensis in the Gulf of Maine

Summary

Studies of juvenile recruitment of the green sea urchin Strongylocentrotus droebachiensis in the Gulf of Maine were conducted during the summer of 1995. These experiments confirmed 12 years of previous observations that settlement only occurs during the months of June and July. Settlement panels were placed at a series of sites along the Maine and New Hampshire coastline to compare recruitment in the northeastern and southwestern regions of the Gulf of Maine. The densities of urchins recruiting in Casco Bay and at the Isles of Shoals were two orders of magnitude higher than those from Eastport and Winter Harbor. There was a discontinuity in settlement densities at Penobscot Bay. Experiments conducted at the Isles of Shoals showed a positive relationship between water motion and larval supply, but neither parameter correlated with recruitment density over eight stations. Contrary to previous results, recuitment was greater within natural, as well as, artificial kelp beds compared to urchin barren areas and control panels outside the experimental kelp beds. The impact of changing community structure due to urchin harvesting was discussed as an factor influencing differences in juvenile urchin recruitment.     

Recruitment and growth of the sea urchin Strongylocentrotus droebachiensis (Muller) following mass mortalities off Nova Scotia,Canada

Recruitment of Strongylocentrotus droebachiensis (Muller) was monitored for four successive years following a mass mortality of this species on a subtidal boulder bottom in St. Margaret's Bay, Nova Scotia. Recently recruited juveniles (1–2 mm diameter) were observed in the fall (September–November) between (1982) and (1984), but not in (1985). Juveniles grew to 6–8 mm diameter at 1-yr post-settlement and 19 mm diameter ((1983) cohort) at 2-yr post-settlement. Individuals > 18 mm diameter had macroscopic gonads in the spring of their third year and spawning probably occurred at 2.75-yr post-settlement. The growth rate of juveniles (3–6 mm initial diameter) maintained in cages with coralline-covered rocks was similar to that of the natural population. Growth and gonad index increased significantly when kelp (Laminaria longicruris (Pylaie)) was added to cages, suggesting that juveniles were food limited in St. Margaret's Bay. Despite an abundance of kelp and other fleshy algae, they remained cryptic under rocks and were not observed grazing on attached plants. The effects of temperature and food supply on juvenile growth and survival were examined in laboratory experiments using factorial designs. Recently-metamorphosed juveniles (from laboratory cultures), maintained at three temperature regimes (5°C, ambient, 16 °C) and two feeding regimes (no added food, coralline algae added), had similar growth rates (over 9 mth) among treatment combinations. However, growth was asymptotic at 16 °C and ambient temperatures, suggesting food limitation. Survival of these juveniles was higher in treatments with coralline algae, but survival was not affected by temperature. Field-collected juveniles (3–6 mm diameter), maintained at three temperature regimes (5°C, ambient, 16 °C) and three feeding regimes (no added food, coralline algae added, and kelp and coralline algae added), grew only in treatments with kelp added as food. In these treatments, growth rates increased with temperature. However, the growth curve at variable ambient temperature regimes was linear, suggesting seasonal acclimation. Survival of the field-collected juveniles was highest in treatments at 5 °C and/or with added kelp.     

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.