In Silico Reconstruction of Sperm Chemotaxis.

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca2+ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. Based on these experimental findings, in this study, we reconstructed a sperm model in silico to demonstrate an algorithm for sperm chemotaxis. We also focused on the importance of desensitizing the chemoattractant receptor in long-range chemotaxis because sperm approach distantly located eggs, and they must sense the chemoattractant concentration over a broad range. Using parameters of the sea urchin, simulations showed that a number of sperm could reach the egg from millimeter-order distances with desensitization, indicating that we could organize a functional sperm model, and that desensitization of the receptor is essential for sperm chemotaxis. Then, we compared the model with starfish sperm, which has a different desensitization scheme and analyzed the properties of the model against various disturbances. Our approach can be applied as a novel tool in chemotaxis research.

Coelomocyte replenishment in adult Asterias rubens: the possible ways.

The origin of cells involved in regeneration in echinoderms remains an open question. Replenishment of circulatory coelomocytes-cells of the coelomic cavity in starfish-is an example of physiological regeneration. The coelomic epithelium is considered to be the main source of coelomocytes, but many details of this process remain unclear. This study examined the role of coelomocytes outside circulation, named marginal coelomocytes and small undifferentiated cells of the coelomic epithelium in coelomocyte replenishment in Asterias rubens. A qualitative and quantitative comparison of circulatory and marginal coelomocytes, as well as changes of circulatory coelomocyte concentrations in response to injury at different physiological statuses, was analysed. The presence of cells morphologically similar to coelomocytes in the context of coelomic epithelium was evaluated by electron microscopy. The irregular distribution of small cells on the surface and within the coelomic epithelium was demonstrated and the origin of small undifferentiated cells and large agranulocytes from the coelomic epithelium was suggested. Two events have been proposed to mediate the replenishment of coelomocytes in the coelom: migration of mature coelomocytes of the marginal cell pool and migration of small undifferentiated cells of the coelomic epithelium. The proteomic analysis of circulatory coelomocytes, coelomic epithelial cells and a subpopulation of coelomic epithelial cells, enriched in small undifferentiated cells, revealed proteins that were common and specific for each cell pool. Among these molecules were regulatory proteins, potential participants of regenerative processes.

Warming and temperature variability determine the performance of two invertebrate predators.

In a warming ocean, temperature variability imposes intensified peak stress, but offers periods of stress release. While field observations on organismic responses to heatwaves are emerging, experimental evidence is rare and almost lacking for shorter-scale environmental variability. For two major invertebrate predators, we simulated sinusoidal temperature variability (±3 °C) around todays' warm summer temperatures and around a future warming scenario (+4 °C) over two months, based on high-resolution 15-year temperature data that allowed implementation of realistic seasonal temperature shifts peaking midpoint. Warming decreased sea stars' (Asterias rubens) energy uptake (Mytilus edulis consumption) and overall growth. Variability around the warming scenario imposed additional stress onto Asterias leading to an earlier collapse in feeding under sinusoidal fluctuations. High-peak temperatures prevented feeding, which was not compensated during phases of stress release (low-temperature peaks). In contrast, increased temperatures increased feeding on Mytilus but not growth rates of the recent invader Hemigrapsus takanoi, irrespective of the scale at which temperature variability was imposed. This study highlights species-specific impacts of warming and identifies temperature variability at the scale of days to weeks/months as important driver of thermal responses. When species' thermal limits are exceeded, temperature variability represents an additional source of stress as seen from future warming scenarios.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica.

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals.

Injury affects coelomic fluid proteome of the common starfish, Asterias rubens.

Echinoderms, possessing outstanding regenerative capabilities, provide a unique model system for the study of response to injury. However, little is known about the proteomic composition of coelomic fluid, an important biofluid circulating throughout the animal's body and reflecting the overall biological status of the organism. In this study, we used LC-MALDI tandem mass spectrometry to characterize the proteome of the cell-free coelomic fluid of the starfish Asterias rubens and to follow the changes occurring in response to puncture wound and blood loss. In total, 91 proteins were identified, of which 61 were extracellular soluble and 16 were bound to the plasma membrane. The most represented functional terms were 'pattern recognition receptor activity' and 'peptidase inhibitor activity'. A series of candidate proteins involved in early response to injury was revealed. Ependymin, ß-microseminoprotein, serum amyloid A and avidin-like proteins, which are known to be involved in intestinal regeneration in the sea cucumber, were also identified as injury-responsive proteins. Our results expand the list of proteins potentially involved in defense and regeneration in echinoderms and demonstrate dramatic effects of injury on the coelomic fluid proteome.

Discriminating Eaters: Sea Stars Asterias rubens L. Feed Preferably on Mytilus trossulus Gould in Mixed Stocks of Mytilus trossulus and Mytilus edulis L.

Sea stars Asterias rubens are important natural enemies of the blue mussel Mytilus in the North Atlantic. We asked whether these predators distinguish between the cryptic species M. edulis and M. trossulus that occur sympatrically in the White Sea. In mixed experimental stocks, the odds of being eaten by sea stars were about four times greater for M. trossulus. We also showed that A. rubens preferred smaller mussels to larger ones, irrespective of their species affinity. Our findings support earlier indirect observations showing that sea stars recognize M. trossulus as a more preferable prey than M. edulis. Dramatic differences in the vulnerability to sea star predation may explain the segregation of habitats between the two mussel species in contact zones; M. trossulus usually tends to occupy habitats where the sea star predators are scarce.

Trans-life cycle acclimation to experimental ocean acidification affects gastric pH homeostasis and larval recruitment in the sea star Asterias rubens.

AIM: Experimental simulation of near-future ocean acidification (OA) has been demonstrated to affect growth and development of echinoderm larval stages through energy allocation towards ion and pH compensatory processes. To date, it remains largely unknown how major pH regulatory systems and their energetics are affected by trans-generational exposure to near-future acidification levels. METHODS: Here, we used the common sea star Asterias rubens in a reciprocal transplant experiment comprising different combinations of OA scenarios, to study trans-generational plasticity using morphological and physiological endpoints. RESULTS: Acclimation of adults to pHT 7.2 (pCO2 3500 µatm) led to reductions in feeding rates, gonad weight and fecundity. No effects were evident at moderate acidification levels (pHT 7.4; pCO2 2000 µatm). Parental pre-acclimation to pHT 7.2 for 85 days reduced developmental rates even when larvae were raised under moderate and high pH conditions, whereas pre-acclimation to pHT 7.4 did not alter offspring performance. Microelectrode measurements and pharmacological inhibitor studies carried out on larval stages demonstrated that maintenance of alkaline gastric pH represents a substantial energy sink under acidified conditions that may contribute up to 30% to the total energy budget. CONCLUSION: Parental pre-acclimation to acidification levels that are beyond the pH that is encountered by this population in its natural habitat (eg, pHT 7.2) negatively affected larval size and development, potentially through reduced energy transfer. Maintenance of alkaline gastric pH and reductions in maternal energy reserves probably constitute the main factors for a reduced juvenile recruitment of this marine keystone species under simulated OA.

Coelomic Transport and Clearance of Durable Foreign Bodies by Starfish (Asterias rubens).

Echinoderms have excellent healing and regeneration abilities, but little is known about how they deal with the related challenge of durable foreign bodies that become lodged within their bodies. Here we report a novel mechanism for foreign body elimination in starfish. When injected into the arm of a starfish, passive integrated transponder tags and magnets of similar dimensions are eliminated at a rate approximating 10% per day. These objects are forcefully ejected through the body wall at the distal tip of an arm. Ultrasound images reveal that foreign bodies are moved within the body cavity, and tracking of magnets injected into starfish suggests that the movements are haphazard rather than directed. Constrictions of the body wall near the foreign object are the likely mechanism for this transport process. Open questions include the ecological relevance of this behavior, why clearance occurs through the distal tips of the arms, the neurological and muscular control of this behavior, what other animals use this mechanism, and the range of objects starfish can eliminate in this way.

Effects of ammonia on fertilization, development, and larval survival in the Northern Pacific asteroid, Asterias amurensis.

For developing a complementary test organism to sea urchin during winter in Korea, sensitivities of sperm, embryo, and larvae of Asterias amurensis to un-ionized ammonia were evaluated. The EC50s (Mean ± SD, n = 3) for fertilization and development were 169 ± 62 and 70 ± 19 µg/L, respectively. The 48, 72, and 96-h LC50s for larval survival were 1,674 ± 583, 498 ± 221, and 336 ± 107 µg/L, respectively. The sensitivities of fertilization, development, and larval survival tests with A. amurensis are higher than or comparable to those of sea urchin and other taxonomic groups. Therefore, fertilization, development, and larval survival tests using A. amurensis are suitable for assessing pore water toxicity of marine sediments in Korea.

Repeatability of escape response performance in the queen scallop, Aequipecten opercularis.

In order for natural selection to operate, physiological and behavioural traits must exhibit both inter-individual variability and intra-individual consistency (i.e. repeatability) in performance. In this study, we describe individual variation and temporal repeatability in the escape responses of the queen scallop, Aequipecten opercularis, and determine whether individuals exhibited consistently high or low rankings in different aspects of the escape response. Five measures of individual performance were recorded on four occasions (days 0, 2, 7 and 28), providing proxies for sensory acuity (response latency), immediate and sustained swimming performance (burst and average clap rates), and swimming endurance (total number of claps and total time spent clapping). All components of the escape response exhibited significant inter-individual variability (all P<0.0001). Escape response latency, burst clap rate, total number of claps and total duration spent clapping maintained significant repeatability over 28 days (all P<0.016). Average clap rate was repeatable in the short term (2 days, P<0.0001) but repeatability declined by 28 days (P=0.097). Concordance analysis indicated that individuals maintained the same performance rankings over time for each component of the escape response (all P<0.001). In addition, some individuals ranked as consistently high or low performers across response latency, burst and average clap rate, and total number of claps. An individual's ability to evade predators through the provision of an escape response of an appropriate magnitude, subject to physiological, behavioural and organismal constraints, will have clear fitness-related consequences.

Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens.

Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named 'Sea star footprint proteins' (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes.

Effects of field contamination by metals (Cd, Cu, Pb, Zn) on biometry and mechanics of echinoderm ossicles.

Echinoderms are known to readily incorporate metals in their calcified endoskeleton. It is currently unclear if this has an impact on the skeleton function or if this can be considered as a detoxification mechanism. In the present work, populations of the sea urchin Echinus acutus and the starfish Asterias rubens were studied in stations distributed along a metal contamination gradient in a Norwegian fjord (Sørfjord). Ossicles involved in major mechanical functions - sea urchin spine and starfish ambulacral plate - were analyzed for their metal concentration (Cd, Cu, Pb and Zn) and their biometric and mechanical properties. Starfish plates were more contaminated by Cd, Pb and Zn than sea urchin spines. Cu concentrations were at background levels. In E. acutus, metals principally affected size. In A. rubens, material stiffness and toughness were decreased in the most contaminated station. This reduction is attributed either to the direct incorporation of metals in the calcite lattice and/or to deleterious effects of metals during skeleton ontogenesis. The contrasting incorporation of metals in the skeleton of the two investigated species accounts for the different impact of the metals, including in terms of fitness. The present results clearly indicate that, at least in A. rubens, incorporation of metals in the skeleton cannot be considered as a detoxification mechanism.

[The characteristic of the coelomic fluid and coelomic epithelium cell populations of starfish Asterias rubens L., capable to attach and spread on various substrates].

Cultivation is one of the methods modeling processes occurring in vivo. The success of cultivation, in particular, is defined by a substratum choice. We studied the ability of coelomocytes and coelomic epithelial cells to attach and spread to fibronectin, laminin, polylysine, and glass. Qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations evaluated in response to injury. Seven relative classes of coelomocytes has been identified, three of which has been shown to participate in the formation of clot during primary repair of wounds. There was a change in the proportion of these cells, attached to specific ligands in response to the injury. In coelomic epithelium 8 relative classes of cells has been identified, two of which are likely to be candidates for the role of progenitor cells for coelomocytes--coelomocyte-like and small epithelial cells with high nuclear-cytoplasmic ratio. The enrichment with the small cells in population of attached coelomic epithelium cells has been revealed when seeding on laminin. Continued viability of epithelial cells has been shown when cultured on laminin during 2 months.

Effects of manganese and hypoxia on coelomocyte renewal in the echinoderm, Asterias rubens (L.).

Manganese (Mn) is a naturally abundant metal and particularly so in soft-bottom oceanic sediments where it generally occurs bound in a four-valent colloidal state as MnO2. When hypoxic conditions occur in bottom waters, the metal reduces to the bioavailable ion Mn2+ and can reach concentrations known to have immunotoxic effects in the crustacean Nephrops norvegicus, reducing numbers of circulating haemocytes as a consequence. However, we have previously shown that Mn seems to have a contrasting effect on the echinoderm Asterias rubens in which it triggers the proliferation of haematopoietic cells and increases coelomocyte numbers. Since elevated Mn levels mostly co-occur with hypoxia in nature, here we investigated whether hypoxia has a negative effect on haematopoiesis. Proliferation and differentiation of coelomocytes and cells in the coelomic epithelium of A. rubens were compared after 3 days of exposure to realistic levels of Mn, hypoxia or a combination of these two parameters. We can confirm that Mn elevated numbers of coelomocytes and increased proliferation of epithelial cells, but hypoxia did not affect these levels. However, hypoxia did affect differentiation of these cells as judged by investigating the expression of a Runt domain transcription factor, which was also cloned and sequenced. Through comparative quantification using a real time PCR technique, we found that exposure to hypoxia had a clearly stimulating effect on mRNA expression of Runt gene in both coelomocytes and epithelial cells. These results indicate that during hypoxic conditions the composition of coelomocyte sub-populations changed.

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L).

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens', was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.

[Cells of coelomic liquid and cells of different tissues of sea star Asterias rubens L. isolated from intact and post-traumatic animals: behaviour and proliferation under cultivation in vitro].

Proposed sources of coelomocytes in Asteroidea after traumatic injures are coelomic epithelium, axial organ or Tidemann's bodies. To study the involvement of cell division in the process, proliferation of cells from different tissues of starfish Asterias rubens L. has been studied after bromdeoxyuridine incorporation in vivo. To study the differentiation of coelomocytes in vitro a method for isolation and cultivation of different tissue cells has been worked out and cell behaviour and proliferation in culture has been analyzed. The reliable BrdU incorporation has been found in coelomic epithelium cells in vivo. Coelomocytes and coelomic epithelium cells behaviour in culture dependent on the post-trauma period after which the cells were loaded into the culture whereas no difference was revealed for axial organ and Tidemann's bodies cells. Two-month cultivation of coelomic epithelium cells resulted in formation of colony-like accumulations of the cells with high nuclear-cytoplasm ratio which of colony-like accumulation of the cells with high nuclear-cytoplasm ratio which incorporated BrdU. Thus, coelomic epithelium cells seem to be more promising object for the study of A. rubens cell differentiation in vitro.

[Ultrastructure of coelomic epithelium and coelomocytes of intact and wounded starfish Asterias rubens L].

Samples of coelomic epithelium and coelomocytes suspension of intact and wounded starfishes Asterias rubens L. were analyzed by electron microcopy. It has been demonstrated that coelomic epithelium is composed of three types of cells: flagellar (approx. 60%), secretory (approx. 3%) and myoepithelial (approx. 37%). Flagellar and secretory cells form the apical surface of coelomic epithelium. Secretory cells are represented by two subtypes: granular and mucous secretory cells. Myoepithelial cells are located in the basal zone of the epithelium. Adjacent flagellar cells are separated by intercellular gaps of various size in 4-5% of cases. These gaps are apparently the lacunae left by the flagellar cells after their departure to the coelomic cavity. The morphological pattern of transition of coelomic epithelium flagellar cells to the coelomocytes has been characterized. No significant structural alterations in organization of the coelomic epithelium were revealed after moderate wounding used in the present study. Small round-shaped young coelomycytes (approx. 3%) and bigger mature coelomocytes (approx. 97%) were found in coelomocytes suspension. A flagellum was revealed on the surface of one of the young coelomocytes. Surface of the mature coelomocytes forms the processes of various size and structure; their cytoplasm contains lysosomes and fagocytic vacuoles of different size. After wounding, activation of coelomocytes was noted finding expression in the sharp rise in the number and the length of their surface filopodia, and in the multicellular aggregates formation. By the sum of the ultrastructural data, histogenesis of coelomocytes from the flagellar cells of the coelomic epithelium is supposed to be a process of cellular transdifferentiation.

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia.

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.

Experimental approaches to evaluating the point of biological system bifurcation.

Any system transforms into a qualitatively new state if the number of its elements changes (increases or decreases in comparison with the standard checkpoint: norm, previous status of the system, etc.) by at least 1/3.