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Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FASTM is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.

The purple-spined sea urchin ( Arbacia punctulata) is commonly found in shallow waters of the western Atlantic Ocean from the New England area of the United States to the Caribbean. Sea urchins play a major role in ocean ecology, echinoculture, and biomedical research. Additionally, sea urchins are commonly displayed in public aquaria. Baseline parameters were developed in unanesthetized urchins for righting reflex (time to regain oral recumbency) and spine response time to tactile stimulus. Tricaine methanesulfonate (MS-222) was used to sedate and anesthetize purple-spined sea urchins and assess sedation and anesthetic parameters, including adhesion to and release from a vertical surface, times to loss of response to tactile stimulus and recovery of righting reflex, and qualitative observations of induction of spawning and position of spines and pseudopodia. Sedation and anesthetic parameters were evaluated in 11 individuals in three circumstances: unaltered aquarium water for baseline behaviors, 0.4 g/L MS-222, and 0.8 g/L MS-222. Induction was defined as the release from a vertical surface with the loss of righting reflex, sedation as loss of righting reflex with retained tactile spine response, anesthesia as loss of righting reflex and loss of tactile spine response, and recovery as voluntary return to oral recumbency. MS-222 proved to be an effective sedative and anesthetic for the purple-spined sea urchin at 0.4 and 0.8 g/L, respectively. Sodium bicarbonate used to buffer MS-222 had no measurable sedative effects when used alone. Anesthesia was quickly reversed with transfer of each individual to anesthesia-free seawater, and no anesthetic-related mortality occurred. The parameters assessed in this study provide a baseline for sea urchin anesthesia and may provide helpful comparisons to similar species and populations that are in need of anesthesia for surgical procedures or research. [ABSTRACT FROM AUTHOR]

Many cells and microorganisms have evolved a motility apparatus to explore their surroundings. For guidance, these biological microswimmers rely on physical and chemical cues that are transduced by cellular pathways into directed movement - a process called taxis. Only few biological microswimmers have been studied as detailed as sperm from sea urchins. Sperm and eggs are released into the seawater. To enhance the chances of fertilization, eggs release chemical factors - called chemoattractants - that establish a chemical gradient and, thereby, guide sperm to the egg. Sea urchin sperm constitute a unique model system for understanding cell navigation at every level: from molecules to cell behaviours. We will outline the chemotactic signalling pathway of sperm from the sea urchin Arbacia punctulata and discuss how signalling controls navigation in a chemical gradient. Finally, we discuss recent insights into sperm chemotaxis in three dimensions (3D). [ABSTRACT FROM AUTHOR]

Many marine organisms have complex life histories, having sessile adults and relying on the planktonic larvae for dispersal. Larvae swim and disperse in a complex fluid environment and the effect of ambient flow on larval behavior could in turn impact their survival and transport. However, to date, most studies on larvae-flow interactions have focused on competent larvae near settlement. We examined the importance of flow on early larval stages by studying how local flowand ontogeny influence swimming behavior in pre-competent larval sea urchins, Arbacia punctulata. We exposed larval urchins to grid-stirred turbulence and recorded their behavior at two stages (4- and 6-armed plutei) in three turbulence regimes. Using particle image velocimetry to quantify and subtract local flow, we tested the hypothesis that larvae respond to turbulence by increasing swimming speed, and that the increase varies with ontogeny. Swimming speed increased with turbulence for both 4- and 6-armed larvae, but their responses differed in terms of vertical swimming velocity. 4-Armed larvae swam most strongly upward in the unforced flowregime, while 6-armed larvae swam most strongly upward in weakly forced flow. Increased turbulence intensity also decreased the relative time that larvae spent in their typical upright orientation. 6-Armed larvae were tilted more frequently in turbulence compared with 4-armed larvae. This observation suggests that as larvae increase in size and add pairs of arms, they are more likely to be passively re-oriented by moving water, rather than being stabilized (bymechanisms associated with increased mass), potentially leading to differential transport. The positive relationship between swimming speed and larval orientation angle suggests that there was also an active response to tilting in turbulence. Our results highlight the importance of turbulence to planktonic larvae, not just during settlement but also in earlier stages through morphology-flow interactions. [ABSTRACT FROM AUTHOR]

The article discusses various studies on chemotaxis in animal sperm. It mentions about a report entitled "The production of sperm isoagglutinins by ova" by F. R. Lillie which described an agglutination reaction when drops of seawater conditioned with unfertilized eggs were injected into a milky suspension of sperm obtained from the sea urchin Arbacia punctulata. It reports about studies which suggest that chemotactic signaling in sperm of marine invertebrates might differ from mammals.

Abstract: In recent decades attention has focused on the development of non-toxic fouling-release coatings based on silicone polymers as an alternative to toxic antifouling coatings. As fouling-release coatings gain market share, they will contribute to environmental contamination by silicones. We report effects of eight model polysiloxane and three commercial foul-release coatings on embryonic development of sea urchins and fish, Japanese medaka. We used model coatings because they have known composition and commercially available components and molecules leaching from these coatings have been partially characterized. The commercial fouling-release coatings are purported to be non-toxic and components are proprietary. Our goal was to expose embryos of well studied model animals to the coatings to determine if the complex mixtures leaching from the coatings impact development. Urchins were chosen because development is rapid and embryos can enter the non-slip layer over surfaces. Medaka was chosen because the female deposits the sticky eggs onto the anal fin and then scrapes them off onto surfaces. Embryos were confined in water over coatings in 24 well plates. Fresh model coatings had no effect on urchin development while commercial fouling-release coatings inhibited development. Fish embryos had delayed hatching, increased mortality of hatchlings and dramatically decreased ability of hatchlings to inflate the swim bladder and reduced hatching success on all coatings. After one-month immersion of coatings in running seawater to simulate initial application in the marine environment, sea urchin embryos died when placed over model silicones. Effects of the commercial coatings were reduced but included retarded development. Effects on fish embryos over leached coating were reduced compared to those of fresh coating and included decreased hatching success, decreased hatchling survival and inability to inflate the swim bladder for commercial coatings. These findings suggest, similar to medical conclusions, compounds leaching from silicone coatings can impact development and the topic deserves study. [Copyright &y& Elsevier]

Abstract: Regular echinoid skeletons, or tests, comprise plate patterns and overall shapes that have proven challenging to analyse solely on the basis of any one approach or process. Herein, we present a computational model, Holotestoid, that emulates four macrostructural ontogenic processes involved in test growth (plate growth, plate addition, plate interaction, and plate gapping). We devise a geometric representation for analysing tests and describe how we use analogies (bubble interactions and close-packing) to emulate the processes. In the computational model, the emulated processes are used to determine the plate size and plate shape and combined to simulate a growth zone. We simulated growth zones for Arbacia punctulata and for Strongylocentrotus franciscanus by changing the value for one parameter, the ambulacral column angle. We quantitatively compared morphological features for simulated forms to those for real specimens to test the computational model. Additionally, we simulated growth zones for A. punctulata, S. franciscanus, Eucidaris thouarsii, and Mellita quinquiesperforata by changing three parameters, ambulacral column angle, peristome radius to apical system radius ratio, and apical system radius to column length ratio. Holotestoid can be used to explain morphological disparity among echinoid tests. [Copyright &y& Elsevier]

The role of echinoid parental nutrition in early-life stage toxicity is not well understood. Arbacia punctulata were fed either a fresh diet consisting of organic lettuce and carrots or a dry feed. Embryos from parents fed the dry feed exhibited lower sensitivity to copper, whereas the opposite occurred with 1,3,5-trinitrobenzene and sodium dodecyl sulfate (SDS). EC50 values for the dry and fresh feed treatments, respectively, were 41.0 and 29.9 μg/L for copper, 0.5 and 1.8 mg/L for 1,3,5-trinitrobenzene, and 3.5 and 5.6 mg/L for SDS. The data suggests that nutritional standardization for sea urchins in ecotoxicological laboratories needs to be addressed and further investigated. [ABSTRACT FROM AUTHOR]

Specific peptides contained within the extracellular layer, or jelly coat, of a sea urchin egg have been hypothesized to play an important role in fertilization, though separate accounting of the effects of chemoattraction, chemokinesis, sperm agglomeration and the other possible roles of the jelly coat have not been reported. In the present study, we used a microfluidic device that allowed determination of the differences in the diffusion coefficients of sperm of the purple sea urchin Arbacia punctulata subjected to two chemoattractants, namely the jelly coat and resact. Our objectives were twofold: (1) to experimentally determine and compare the diffusion coefficients of Arbacia punctulata spermatozoa in seawater, jelly coat solution and resact solution; and (2) to determine the effect of sea urchin sperm diffusion coefficient and egg size on the sperm-egg collision frequency using stochastic simulations. Numerical values of the diffusion coefficients obtained by diffusing the spermatozoa in seawater, resact solution and jelly coat solution were used to quantify the chemotactic effect. This allowed direct incorporation of known enlargements of the egg, and altered sperm diffusion coefficients in the presence of chemoattractant, in the stochastic simulations. Simulation results showed that increase in diffusion coefficient values and egg diameter values increased the collision frequency. From the simulation results, we concluded that type of sperm, egg diameter and diffusion coefficient are significant factors in egg fertilization. Increasing the motility of sperm appears to be the prominent role of the jelly coat. [ABSTRACT FROM AUTHOR]

Abstract: Cellular cytotoxicity is a key component of animal innate immune responses that is one of the first lines of defense against invaders. There is increasing interest in the study of the cellular immune response, particularly non-specific cytotoxic cells and natural killer cells and their receptors. Studies of non-specific cytotoxic cell and natural killer cell recognition and killing (and the receptors involved) will reveal new and important insights into cellular mechanisms of host defense. Here we describe mAbs specific for coelomocyte sub-populations of the purple sea urchin, Arbacia punctulata, using highly purified coelomocyte populations as the antigen source. Monoclonal antibodies were selected using flow cytometric screening methods. Several of the mAbs were shown to bind to two sub-types of coelomocytes when assayed by fluorescence microscopy. Furthermore, these mAbs inhibited coelomocyte cytotoxicity against vertebrate target cells in a functional assay. The mAbs have been used in immunoprecipitation studies. [Copyright &y& Elsevier]

The influence of potentially confounding factors has been identified as a concern for interpreting sea urchin porewater toxicity test data. The results from >40 sediment-quality assessment surveys using early–life stages of the sea urchin Arbacia punctulata were compiled and examined to determine acceptable ranges of natural variables such as pH, ammonia, and dissolved organic carbon on the fertilization and embryological development endpoints. In addition, laboratory experiments were also conducted with A. punctulata and compared with information from the literature. Pore water with pH as low as 6.9 is an unlikely contributor to toxicity for the fertilization and embryological development tests with A. punctulata. Other species of sea urchin have narrower pH tolerance ranges. Ammonia is rarely a contributing factor in pore water toxicity tests using the fertilization endpoint, but the embryological development endpoint may be influenced by ammonia concentrations commonly found in porewater samples. Therefore, ammonia needs to be considered when interpreting results for the embryological development test. Humic acid does not affect sea urchin fertilization at saturation concentrations, but it could have an effect on the embryological development endpoint at near-saturation concentrations. There was no correlation between sediment total organic carbon concentrations and porewater dissolved organic carbon concentrations. Because of the potential for many varying substances to activate parthenogenesis in sea urchin eggs, it is recommended that a no-sperm control be included with every fertilization test treatment. [ABSTRACT FROM AUTHOR]

Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca2+ concentration in the flagellum, which control the swimming behaviour during chemotaxis. The signalling pathway downstream of the synthesis of cGMP by a guanylyl cyclase is ill-defined. In particular, the ion channels that are involved in Ca2+ influx and their mechanisms of gating are not known. Using rapid voltage-sensitive dyes and kinetic techniques, we record the voltage response that is evoked by the chemoattractant in sperm from the sea urchin Arbacia punctulata. We show that the chemoattractant evokes a brief hyperpolarization followed by a sustained depolarization. The hyperpolarization is caused by the opening of K+-selective cyclic-nucleotide-gated (CNG) channels in the flagellum. Ca2+ influx commences at the onset of recovery from hyperpolarization. The voltage threshold of Ca2+ entry indicates the involvement of low-voltage-activated Cav channels. These results establish a model of chemosensory transduction in sperm whereby a cGMP-induced hyperpolarization opens Cav channels by a 'recovery-from-inactivation' mechanism and unveil an evolutionary kinship between transduction mechanisms in sperm and photoreceptors. [ABSTRACT FROM AUTHOR]

The chronic toxicity of silver to the sea urchin (Arbacia punctulata) was determined in 30‰ salinity seawater during a three-part study: A fertilization test (1-h sperm exposure), a 48-h embryo test, and a 30-d adult test. Combined data from the three tests resulted in a lowest-observed-effect concentration of 19 µg/L, a no-observed-effect concentration of 8.6 µg/L, and a maximum acceptable toxicant concentration of 13 µg/L, based on measured concentrations of dissolved silver. The 96-h median effective concentration was 40 µg/L, and the acute to chronic toxicity ratio was 3.1. During the tests, measured concentrations of free ionic silver (Ag+) were only 0.0027 to 0.0046% of dissolved silver concentrations, as predicted by ion-speciation theory. Some measured Ag+ concentrations were lower than predicted, indicating the presence of other ligands in the seawater test media. These strong sulfide ligands were exuded by the exposed sea urchins into the seawater (where Ag-sulfide complexes formed) in amounts that increased in direct proportion to the silver concentration during the toxicity test. This suggests a toxicity-defense mechanism that functioned by modifying the chemistry of the surrounding external medium. [ABSTRACT FROM AUTHOR]

The first investigation of seaweed chemical defense against herbivores involved the brown seaweed Fucus vesiculosus and suggested defense via phlorotannins. The first demonstration of seaweed induction of secondary metabolites in response to herbivory also involved the genus Fucus and assumed a defensive function for phlorotannins. Many other investigations correlate herbivore feeding preference with changing levels of phlorotannins in this genus and others, but few directly test the effects of phlorotannins. No studies have assessed Fucus chemical defenses using bioassay-guided separation to investigate the complete complement of compounds deterring herbivores. We investigated the deterrence of F. vesiculosus chemical extracts using herbivore bioassays to guide our chemical investigations. Although crude extracts from F. vesiculosus strongly deterred feeding by the sea urchin Arbacia punctulata, phlorotannins from this extract did not deter feeding at 2× or 4× natural concentration by dry mass. Feeding deterrence was due to: (1) a polar galactolipid in the ethyl acetate-soluble extract, and (2) a non-phenolic compound, or compounds, in the water-soluble extract. Although this is the first evidence of galactolipids deterring herbivores, such defenses could be geographically and taxonomically widespread. The galactolipid we discovered in Fucus occurs in marine dinoflagellates, and a related metabolite that deters herbivory has recently been discovered in a tropical green seaweed. We were unable to identify the second deterrent compound, but deterrence occurred in a fraction containing carbohydrates, including sulfated sugars, but no phlorotannins. Given the polarity of these chemical deterrents, they could co-occur with and confound bioassays of phlorotannins if investigators test phlorotannin-containing algal extracts without further purification. [ABSTRACT FROM AUTHOR]

One of the most fundamental questions of fertilization is how do sperm locate eggs? In many animals and lower plant groups, sperm are guided by chemo-attractants released from eggs. In this issue of Nature Cell Biology, Kaupp and colleagues now examine the very early events of this process in the sea urchin Arbacia punctulata. [ABSTRACT FROM AUTHOR]

Various protein kinases are activated in eggs in response to fertilization. We have previously shown that the induction of DNA-dependent protein phosphorylation activity in the sea urchin eggs is triggered by fertilization. The present study demonstrates that the activation of a DNA-dependent serine/threonine kinase in unfertilized eggs of Arbacia punctulata can be achieved without fertilization. Prolonged incubation in seawater resulted in the activation of the eggs with concomitant induction of DNA-dependent protein phosphorylation activity. The activated eggs when fertilized show a slight increase in the phosphorylation activity 10-min post-insemination. The activity gradually declines as the first and second cleavages proceed. The cytoplasmic extracts of the blastulae, gastrulae, and plutei lack the enzyme activity. These findings reveal that not only fertilization but also egg activation serves as a signal for the induction of a DNA-dependent protein phosphorylation activity in sea urchin eggs suggesting that sperm-entry is not required for the induction of the enzyme activity. [Copyright &y& Elsevier]

A toxicity database for ordnance compounds was generated using eight compounds of concern and marine toxicity tests with five species from different phyla. Toxicity tests and endpoints included fertilization success and embryological development with the sea urchin Arbacia punctulata; zoospore germination, germling length, and cell number with the green macroalga Ulva fasciata; survival and reproductive success of the polychaete Dinophilus gyrociliatus; larvae hatching and survival with the redfish Sciaenops ocellatus; and survival of juveniles of the opossum shrimp Americamysis bahia (formerly Mysidopsis bahia). The studied ordnance compounds were 2,4- and 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2,4,6-trinitrophenylmethylnitramine (tetryl), 2,4,6-trinitrophenol (picric acid), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The most sensitive toxicity test endpoints overall were the macroalga zoospore germination and the polychaete reproduction tests. The most toxic ordnance compounds overall were tetryl and 1,3,5-trinitrobenzene. These were also the most degradable compounds, often being reduced to very low or below-detection levels at the end of the test exposure. Among the dinitro- and trinitrotoluenes and benzenes, toxicity tended to increase with the level of nitrogenation. Picric acid and RDX were the least toxic chemicals tested overall. [ABSTRACT FROM AUTHOR]

Presents information on a study which estimated the degree to which maternal energy is invested in the jelly coats surrounding eggs of the echinoid Arbacia punctulata. Overview of the structure of echinoid eggs and jelly coats; Implications of investment of energy in jelly coats; Methodology and results; Analysis of results.

Presents information on a study which calculated the shear stress experienced by eggs in the oviduct of the echinoid Arbacia punctulata during spawning using engineering equations that describe the luminar flow through pipes. Materials and methods of the study; Results and discussion.

The article presents a study that examines the effect of protracted cadmium exposure on the viability of eggs and sperm of sexually mature purple sea urchin, Arbacia punctulata. Results show that exposure of sexually mature male and female sea urchins to sea water with solute cadmium could affect the ability of sea urchin gametes to produce normal larva, which is noted to be in greater degree among male gametes. From these results, it has been suggested that cadmium can affect and alter embryotic development through the exposure of parents even at lower concentrations.

The article presents a study on the evaluation of the toxicity of antiscalant agents on the embryos and gametes of Arbacia punctualata. The study uses various methods such as examining the effects of polyacrylamide on the sperm and embryo respiration, and evaluating how the polymers affects the development of the embryos. Results show decrease on respiration rates of sea urchin treated with polyacrylamide levels, and the identical abnormalities of embryos treated with polymers.

An associational plant refuge occurs when a plant that is susceptible to herbivory gains protection from herbivory when it is associated with another plant. In coastal North Carolina, the abundance of the palatable red alga Gracilaria tikvahiae is positively correlated with the abundance of the unpalatable brown alga Sargassum filipendula during times of increased herbivore activity. To see if grazing by the sea urchin Arbacia punctulata could generate this pattern, controlled experiments were conducted in out-door microcosms and in the laboratory. Gracilaria beneath a canopy of Sargassum was eaten significantly less than Gracilaria alone. When Arbacia were excluded, Gracilaria alone grew significantly more than Gracilaria beneath Sargassum, demonstrating that Sargassum is a competitor of Gracilaria. Experiments investigating Sargassum's deterrent role indicated that Sargassum decreased the foraging range of Arbacia and the rate at which it fed on Gracilaria. Additional experiments with plastic Sargassum mimics indicated that the decreased grazing on Gracilaria was not a result of Sargassum morphology, but was probably attributable to some chemical characteristic of Sargassum. The pattern of increased grazing in monocultures (only Gracilaria present) versus polycultures (both Gracilaria and Sargassum present) demonstrated in this study also has been demonstrated for plant-insect interactions in terrestrial communities. In these communities, insect density is higher in monocultures than in polycultures because insects find and immigrate to monocultures more rapidly, and once in a monoculture, they emigrate from them less often than from polycultures. In this study, urchins did not find and immigrate to monocultures more rapidly, nor did they tend to stay in them once they were found; in fact, they emigrated from monocultures of Gracilaria more rapidly than from Gracilaria and Sargassum polycultures. Increased grazing in Gracilaria monocultures resulted from increased rates of movement and feeding of individual herbivores, not from increased herbivore density as has been reported for terrestrial systems. [ABSTRACT FROM AUTHOR]

Features a study which examined the direct effects of nitric oxide on the development of the sea urchin Arbacia punctulata. Effects of a range of concentrations of S-nitroso-N-acetylpenicillamine (SNAP); Application of SNAP after the fertilization of sea urchins.

The article focuses on a study by Karen Chan and colleagues regarding orientation of pre-competent sea urchin Arbacia punctulata in turbulence and ontogenetic changes in larval swimming, that appeared in the journal "Journal of Experimental Biology". It mentions the increase in turbulence of larvae that provide tumbling motions. It also mentions reduction in stability due to larvae acquire more arms.

Discusses research on whether increases in intracellular calcium and motility in the sperm of the sea urchin Arbacia punctulata that are mediated by egg-derived products are also mediated by ryanodine-gated intracellular calcium stores. Results of the experiments; Regulation of ryanodine receptors; Details of a schematic representation of events leading to the activation of Arbacia punctulata sperm.

Part I. Features a study which examined the fertilization of stratified eggs of the sea urchin Arbacia punctulata observed under a centrifuge polarizing microscope. Responses of unfertilized stratified eggs to the calcium ionophore.

Investigates the effects of a structural analog of L-arginine, NG-monomethyl-L-arginine (NMMA) on the fertilization process using eggs from the sea urchin Arbacia punctulata. Analysis of hydrogen peroxide (H2O2) production induced during fertilization using fluorescence imaging; Catalase activity in sperm and eggs; NMMA effect on H2O2 formation during fertilization and development.

The article presents a study which explains that the sperms of the sea urchin specie Arbacia punctulata have a unique navigational pattern leading to the eggs. Researchers Nachiket Kashikar, U. Benjamin Kaupp, and colleagues have noted a gradient of chemical attractants, the peptide resact, that sticks to the receptors on the flagellum creating an in influx of calcium. Moreover, the surges in calcium indicate for the sperm to make sharp and gentle turns in a circuitous path to the egg.

In many benthic communities predators play a crucial role in the population dynamics of their prey. Surface characteristics of the prey are important for recognition and handling by the predator. Because the establishment of an epibiotic assemblage on the surface of a basibiont species creates a new interface between the epibiotized organism and its environment, we hypothesised that epibiosis should have an impact on consumer-prey interactions. In separate investigations, we assessed how epibionts on macroalgae affected the susceptibility of thelatter to herbivory by the urchin Arbacia punctulata and how epibionts on the blue mussel Mytilus edulis affected its susceptibility to predation by the shore crab Carcinus maenas. Some epibionts strongly affected consumer feeding behavior. When epibionts were more attractive than their host, consumer pressure increased. When epibionts were less attractive than their host or when they were repellent, consumer pressure decreased. In systems that are controlled from the top-down,epibiosis can strongly influence community dynamics. For the Carcinus/Mytilus system that we studied, the in situ distribution of epibionts on mussels reflected the epibiosis-determined preferences of the predator. Both direct and indirect effects are involved in determiningthese epibiont-prey-consumer interactions. [ABSTRACT FROM AUTHOR]