Starfish Marthasterias Glacialis Research Articles

More than a simple epithelial layer: multifunctional role of echinoderm coelomic epithelium

In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. However, its overall functions have been scarcely studied in detail. In this work, we used an integrated approach based on both microscopy (light and electron) and proteomic analyses to investigate the arm CE in the starfish Marthasterias glacialis during different physiological conditions (i.e., non-regenerating and/or regenerating). Our results show that CE cells share both ultrastructural and proteomic features with circulating coelomocytes (echinoderm immune cells). Additionally, microscopy and proteomic analyses indicate that CE cells are actively involved in protein synthesis and processing, and membrane trafficking processes such as phagocytosis (particularly of myocytes) and massive secretion phenomena. The latter might provide molecules (e.g., immune factors) and fluids for proper arm growth/regrowth. No stem cell marker was identified and no pre-existing stem cell was observed within the CE. Rather, during regeneration, CE cells undergo dedifferentiation and epithelial-mesenchymal transition to deliver progenitor cells for tissue replacement. Overall, our work underlines that echinoderm CE is not a “simple epithelial lining” and that instead it plays multiple functions which span from immunity-related roles as well as being a source of regeneration-competent cells for arm growth/regrowth.

Characterization of Soluble Cell-Free Coelomic Fluid Proteome from the Starfish Marthasterias glacialis.

Proteomics combined to advanced bioinformatics tools is acquiring a pivotal role in the comprehensive understanding of living organism's biology, in particular for non-model organisms, which includes most marine and aquatic invertebrates. Depicting of protein composition in a whole organ/organism followed by their assembling in functional protein association networks promotes the understanding of key biological processes. Here, we provide a detailed description of the extraction procedure of cell-free coelomic fluid soluble proteins and the characterization of the proteome of the starfish Marthasterias glacialis. Due to coelomic fluid richness in glycoproteins, which complicates protein identification, extracts of soluble proteins are deglycosylated prior to tandem mass spectrometry. This experimental approach is useful at improving knowledge on the coelomic fluid physiological role and deciphering its involvement in regeneration of starfish body parts when comparing different regeneration conditions.

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach.

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

Size-dependent predation of the mesopredator Marthasterias glacialis (L.) (Asteroidea)

Asteroids are largely recognized as important predators in all of the world’s oceans and for this reason, they play a crucial role in shaping the structure and functioning of benthic ecosystems. The spiny starfish Marthasterias glacialis is generally considered a voracious predator of molluscs, in particular bivalves. Using field observations and carbon (δ13C) and nitrogen (δ15N) stable isotopes, we explored possible changes in diet in relation to size of M. glacialis. Data were collected at Ustica Island (Southern Tyrrhenian Sea) from June 2008 to June 2010. M. glacialis showed a size shift in feeding preferences due to different use of food items: bivalves, Columbella rustica, Euthria cornea and Trochoidea were strongly selected by small starfish whereas the larger ones preferred Arbacia lixula and Paracentrotus lividus. The clear picture that emerged is that large M. glacialis is able to control sea urchin populations and indirectly Mediterranean rocky reef ecosystems.

Proteolytic events are relevant cellular responses during nervous system regeneration of the starfish Marthasterias glacialis

The molecular pathways that trigger the amazing intrinsic regenerative ability of echinoderm nervous system are still unknown. In order to approach this subject, a 2D-DIGE proteomic strategy was used, to screen proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. A total of 528 proteins showed significant variations during radial nerve cord regeneration in both soluble and membrane protein-enriched fractions. Several functional classes of proteins known to be involved in axon regeneration events in other model organisms, such as chordates, were identified for the first time in the regenerating echinoderm nervous system. Unexpectedly, most of the identified proteins presented a molecular mass either higher or lower than expected. Such results suggest a functional modulation through protein post-translational modifications, such as proteolysis. Among these are proteins involved in cytoskeleton and microtubule regulators, axon guidance molecules and growth cone modulators, protein de novo synthesis machinery, RNA binding and transport, transcription factors, kinases, lipid signaling effectors and proteins with neuroprotective functions. In summary, the impact of proteolysis during regeneration events is here shown, although requiring further studies to detail on the mechanisms involving this post-transcriptional event on nervous system regeneration. The nervous systems of some organisms present a complete inability of neurons to regrow across a lesion site, which is the case of the adult mammalian central nervous system (CNS). Expanding our knowledge on how other animals regenerate their nervous system offers great potential for groundbreaking biomedical applications towards the enhancement of mammalian CNS regeneration. In order to approach this subject, a 2D-DIGE proteomic strategy was used for the first time, to screen the proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. We strongly believe in the relevance of our results and have clear evidences that this work constitutes a solid basis for new research on starfish regenerating nerve cord. We also believe this work will have a significant impact not only on the general scientific community as we present here an alternative animal model to neurobiology, but also on the scientific community that works with echinoderms or closely related marine invertebrates, which are constantly searching for specific protein markers of several tissues, thus constituting an important advance towards the improvement of large scale protein information of unsequenced, but yet not less important organisms.

Localization of the SALMFamide neuropeptides in the starfish Marthasterias glacialis

In echinoderms, the SALMFamide neuropeptides sharing the SxL/FxFamide motif seem widespread throughout the phylum and may be important signalling molecules that mediate various physiological functions. Recent identification of S1 and its analogues, MagS3 and MagS4, along with the S2 analogue, MagS2 from the starfish Marthasterias glacialis, indicated that SALMFamides in the class Asteroidea are more diverse than previously thought. Further, isolation of the neuropeptides from the radial nerve cord and studies on pharmacological actions of the neuropeptides on the cardiac stomach warrant studies on the tissue distributions of these peptides in both the nervous and digestive systems. In the present study, antisera raised against an S1 analogue, KYSALMFamide, and an S2 analogue, KYSGLTFamide, were used to localize the distribution patterns of the S1- and S2-like immunoreactivities (S1-IR/S2-IR) in the nervous and digestive systems of the starfish. In the nervous system, cell bodies in the ectoneural part were immunostained for both S1 and S2 peptides, while in the digestive system, the basi-epithelial plexus and mucosal cell bodies were immunoreactive. These immunocytochemical data support the notion that the SALMFamides may play a neuroendocrine role in mediating feeding behaviour of the starfish. Further studies including identification of peptide binding sites and differential expression pattern of mRNAs encoding the peptides are required to elucidate their physiological functions.

Role of food availability in the bathymetric distribution of the starfish <i>Marthasterias glacialis</i> (Lamk.) on reefs of northern Portugal

SUMMARY: We examined whether the abundance and size of the starfish Marthasterias glacialis (Lamk.) exhibit a depthdependent partitioning on subtidal reefs. We tested the hypothesis that differences in food availability can result in habitat partitioning along a depth gradient. The abundance and size of M. glacialis was registered at 4 depth strata: 0-4 m, 4-8 m, 8-12 m, and >12 m; we also recorded the number of food items that they were preying on. The abundance and size of M. glacialis decreased with depth. Mussels (Mytilus galloprivincialis) were the most preyed food item across all depth strata, followed by gastropods, sea urchins and barnacles; M. glacialis also consumed a significantly larger amount of mussels in feeding experiments compared with sea urchins and gastropods. The abundance of M. galloprivincialis beds decreased with depth. The clear link between the decrease in abundance and size of M. glacialis with depth and the decay of the most consumed prey (mussels) suggest that food availability may play an important role in the vertical distribution of this starfish, though wave-associated turbulence in the first few metres of the subtidal could also limit the abundance of M. glacialis.

Investigation of benthic community change over a century-wide scale in the western English Channel

Since the early part of the 20th Century the impact of a range of anthropogenic activities in our coastal seas has steadily increased. The effect of such activities is a major cause for concern but in the benthic environment few studies exist that date back more than a few decades. Hence understanding long term changes is a challenge. Within this study we utilized a historic benthic dataset and resurveyed an area west of Eddystone reef in the English Channel previously investigated 112 years ago. The aim of the present work was to describe the current benthic community structure and investigate potential differences between 1895 and 2007. For each of the four major phyla investigated (Polychaeta, Crustacea, Mollusca and Echinodermata), multivariate community analysis showed significant differences between the historic and contemporary surveys. Echinoderm diversity showed a clear reduction between 1895 and 2007. The sea urchins Echinus esculentus, Spatangus purpureus, and Psammechinus miliaris and large star-fish Marthasterias glacialis showed reductions in abundance, in some cases being entirely absent from the survey area in 2007. Polychaetes showed a shift from tubiculous species to small errant and predatory species such as Glycera, Nephtys, and Lumbrineris spp. Within the group Mollusca large species such as Pecten maximus and Laevicardium crassum decreased in abundance while small species increased. Crustaceans in 1895 were dominated by crab species which were present in similar abundances in 2007, but, the order Amphipoda appeared to show a significant increase. While some of the differences observed could stem from differences in methodologies between the surveys, in particular increases of small cryptic species, the loss of large conspicuous species was judged to be genuine. The study area is an important beam trawling and scallop dredging ground; the differences observed are concomitant with changes generally associated with disturbance from demersal fishing activities such as these.

Fish versus starfish predation in controlling sea urchin populations in Mediterranean rocky shores

In the Mediterranean, fishing bans generally allow the recovery of populations of sea urchin predators, such as the seabreams Diplodus sargus and D. vulgaris, promoting the transforma- tion of overgrazed communities into ones dominated by erect macroalgae. However, in the marine reserve on Ustica Island (SW Italy) the opposite trend has occurred in the upper infralittoral commu- nity, and urchin barrens formed after the cessation of fishing activities. We hypothesized that (1) the natural scarcity of the 2 seabream species leads to a low predation rate on sea urchins at Ustica, and (2) predation rate varies with depth, due to differences in the predator assemblages. Tethering experiments were conducted to test these hypotheses by comparing the predation rates at different depths at Ustica and 2 other locations in Sicily characterized by erect macroalgae communities and low urchin densities. Differences in the assemblages of predators, relative vulnerability of the 2 spe- cies of sea urchin present (Paracentrotus lividus and Arbacia lixula), and sea urchin density were also assessed. Despite large variation in predator abundance among locations, there was no difference in predation rate on tethered urchins. Predation by fish was found to be higher in locations where seabreams were abundant, while predation by the starfish Marthasterias glacialis, usually consid- ered a minor sea urchin predator, was found to be the major source of mortality at Ustica. The paucity of predatory fish at Ustica was consistent with the high abundances of urchins and persistence of barrens habitat. The high predation rate on urchins by M. glacialis at Ustica suggests that this species might play an important role in controlling urchin populations in the future.

Identification of novel SALMFamide neuropeptides in the starfish Marthasterias glacialis

The SALMFamides are a family of neuropeptides found in species belonging to the phylum Echinodermata and which act as muscle relaxants. The first two members of this family to be identified were both isolated from the starfishes Asterias rubens and Asterias forbesi and are known as S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide). However, little is known about the occurrence and characteristics of SALMFamide neuropeptides in other starfish species. Here we report the identification of four SALMFamide neuropeptides in the starfish Marthasterias glacialis: GFNSALMFamide (S1), SGPYSMTSGLTFamide (MagS2), AYHSALPFamide (MagS3), and AYQTGLPFamide (MagS4). Analysis of the effects of MagS2 and MagS3 on cardiac stomach preparations from Asterias rubens revealed that both peptides cause dose-dependent relaxation, consistent with previous studies using S1 and S2. The identification of four SALMFamide neuropeptides in Marthasterias glacialis provides new insights into the diversity and phylogenetic distribution of SALMFamide neuropeptides in the class Asteroidea of the phylum Echinodermata. In particular, the identification of MagS3 and MagS4, in addition to S1 and the S2-like peptide MagS2, has revealed a greater diversity of SALMFamide neuropeptides occurring in a starfish species than any previous studies.

Sponge Coating Decreases Predation On The Bivalve Arca Noae

Mediterranean populations of the bivalve Arca noae were found to be specifically fouled by the demosponge Crambe crambe. The surface of the shells forms spines which favour settlement of the sponge larvae. The reproductive strategy of this symbiotic sponge contrasts with other Mediterranean sponges, which produce numerous small larvae, while Crambe crambe produces relatively small numbers of quite large larvae containing secondary metabolites. The selective shell fouling of Arca noae suggests that this is behavioural farming of a chemically defended sponge by a bivalve which does not sequester or synthesize de novo metabolites. Some of the observed benefits of this symbiosis are the enhanced survival of the bivalve, and the sponge being able to live in a site free of competitors. This mutual symbiosis is optional for the sponge but essential for the bivalve, since the fouled bivalves can use the secondary metabolites produced by the sponge to deter predators. We tested the effects of sponge epibiosis on predator preferences. Feeding trials were carried out on the starfish Marthasterias glacialis and the snail Hexaplex trunculus in Hornillo Bay, SE Spain. When A. noae fouled by the sponge Crambe crambe were offered to starfish and snails, they survived significantly more than unfouled bivalves. Field studies in the Mar Menor lagoon indicate that the aposymbiotic populations of A. noae have disappeared from this hypersaline lagoon due to the biological invasion by the non-native snail Hexaplex trunculus. This symbiotic relationship was also effective against octopus. Aposymbiotic bivalves were rapidly eaten when presented to octopuses, while fouled bivalves were consistently rejected.

Anti-adhesive glycoproteins in echinoderm mucus secretions

Marine invertebrates produce a large variety of mucus secretions which are rich in glycoproteins. As part of our studies of natural antifouling mechanisms, mucus secretions from the starfish Marthasterias glacialis and Porania pulvillus and the brittlestar Ophiocomina nigra have been used to characterise the structure and function of some of the glycoproteins present in these secretions. Mucus was collected from all three species and fractionated by size exclusion chromatography. A high molecular weight glycoprotein fraction was collected from each species. Monosaccharide analysis and FTIR demonstrated a composition consistent with a mucin-type glycoprotein. The mucin from M. glacialis and O. nigra inhibited in vitro bacterial adhesion in a dose-dependent manner. In contrast, the mucin from P. pulvillus promoted bacterial adhesion in a dose-dependent manner. All of the mucins inhibited the adhesion of human neutrophils to cultured human vascular endothelial cells (HUVECs) and had no anticoagulant activity. The mucins described here have adhesion-regulating functions that may have a role in the antifouling or feeding mechanisms of the organisms that produce them. These mucins may also be of therapeutic value through their ability to regulate human neutrophil adhesion or bacterial adhesion.

Synthesis of 3β,6α-dihydroxy-5α-cholan-23-one

Evidence for the presence of 3β,6α-dihydroxy-5α-chol-9(11)-en-23-one in the aglycone mixture from the starfish Marthasterias glacialis is provided by the synthesis of 3β,6α-dihydroxy-5α-cholan-23-one ( 19 ) and its identification in the hydrogenated aglycone mixture. The side-chain is constructed from the 23,24-dinorcholanol ( 13 ) by reaction of the 22-tosylate ( 16 ) with the acetylide anion, followed by hydration of the resulting 23-yne ( 17 ).

A link between predator density and arm loss in the starfish Marthasterias glacialis and Asterias rubens

Arm loss in two species of starfish Marthasterias glacialis and Asterias rubens (Echinodermata: Asteroidea) was recorded at eight sites in Lough Hyne, south-west Ireland. There was a significant positive correlation between the density of predators (Luidia ciliaris (Echinodermata: Asteroidea) and Cancer pagurus (Crustacea: Brachyura)) and arm loss in both species of starfish. The results suggest that predators may be responsible for a significant proportion of observed arm loss in these two species of starfish in Lough Hyne.

Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis

The neuroendocrine system of the starfish Marthasterias glacialis was investigated immunocytochemically using antisera specific for rat neuronal, bovine aortic endothelial, and mouse macrophage, nitric oxide (NO) synthases. Immunoreactivity was detected only with the antibodies specific for the neural enzyme, in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract. The pyloric stomach showed more immunoreactive structures than the other digestive organs, with the rectal caeca showing the least activity. Immunoreactive endocrine cells were located in the cardiac and pyloric stomachs and in the pyloric caeca. Co-localization of the enzyme immunoreactivity, and the staining for NADPH-diaphorase, demonstrate the presence of NO synthase in echinoderms. These results provide further evidence that NO is a neuronal messenger of early phylogenetic origin which has been conserved throughout evolution.

Ultrastructural Histochemistry of Marthasterias glacialis (Echinodermata, Asteroidea) Gametes Before and After Fertilization.

The gametes of the starfish Marthasterias glacialis were studied, both before and after fertilization, using several histochemical methods at the ultrastructural level. Results showed that the oocyte jelly coat (JC) could be morphologically differentiated into a fibrillar network of acidic polysaccharides, probably containing glycosaminoglycans (GAGs), and a loose component made of neutral polysaccharides that occupies the interstices of the former. Results also suggested that it is the fibrillar component of the JC to which the spermatozoon attaches and reacts. Staining of the acrosomal vesicle contents confirmed that the peripheral dense component attaches the spermatozoon to the JC, whereas the central and apical electron-lucent component coats the acrosomal process as it extends through the oocyte investments. The acrosomal process membrane was also shown to be negatively charged. Staining of cortical vesicles confirmed their tripartite compartmentalization: the spiral lamellae fused with the fertilization envelope (FE), transferring its staining to the inner layer of the FE, whereas the matrix could be subdivided into two components--a fibrillar mesh of acidic polysaccharides that fused with the FE and some amorphous aggregates that seem to become dispersed in the perivitelline space.

Regulatory peptides in gut endocrine cells and nerves in the starfish Marthasterias glacialis

The endocrine cells of the starfish digestive tract are spindle-shaped, contacting both the lumen and the basiepithelial plexus. Silver impregnation labels the basiepithelial and subcoelomic plexuses as well as these cells. Twenty antisera have been tested using the avidinbiotin method, in order to identify the regulatory substances involved in this system. Endocrine cells and nerves immunoreactive to GFNSALMFamide- (S1), FMRFamide-, peptide tyrosine-tyrosine-(PYY), pancreatic polypeptide- (PP), melanocyte stimulating hormone- (αMSH) and peptidylglycine alpha-amidating monooxygenase- (PAM) specific antisera have been found in the epithelium. The antibodies against S1, a peptide isolated from the nervous system of a starfish, and αMSH, stain both the basiepithelial plexus and the subcoelomic plexus, but the others react only with nerves in the basiepithelial plexus. Absorption controls show that antibodies for S1 and FMRFamide totally crossreact recognizing the same molecule, possibly S1. The other antibodies do not show cross-reactivity to any of the rest, and thus we conclude that these regulatory peptides are present in starfish. This is the first report of the presence of FMRFamide, PYY, αMSH and PAM in the Echinodermata. Under the electron microscope the endocrine cells exhibit secretory granules, microtubules and mitochondria. Direct contact with the subcoelomic plexus can be observed.

Silver staining of spermatogenesis in the starfishMarthasterias glacialis

Summary Spermatogenesis of the starfish Marthasterias glacialis was studied by electron microscopy with the application of the silver nitrate method. In the nucleus, silver stained the nucleolus and specific regions at the periphery of chromatin. In the cytoplasm, silver deposits appear as argyrophilic bodies, on midpiece and tail structures, and on the dense materials which line the intercellular bridges and the inner acrosomal vesicle membrane.

Starfish acrosomal acid phosphatase: a cytochemical and biochemical study

The acrosome reaction of spermatozoa from the starfish Marthasterias glacialis was induced with the ionophore A23187. Reacted cells were then processed for acid phosphatase ultrastructural cytochemistry, but significant enzyme activity was not detected. However, when the supernates from suspensions of ionophore-treated sperm were assayed for acid phosphatase, a net enzyme activity was observed. Supernatant proteins were run in starch gel electrophoresis and fluorescent zymograms revealed a single band of acid phosphatase. SDS-PAGE of proteins eluted from the active spots of starch gels showed one major band of about 63 kDa. The results obtained support the hypothesis that the acid phosphatase whose activity has been detected only at the time of binding of sperm and egg originates from the sperm acrosome.

Fine structural study of the acrosome formation in the starfish Marthasterias glacialis (Echinodermata, Asteroidea)

The fine structure of the spermatogenic cells in the starfish Marthasterias glacialis was studied regarding acrosome formation. The main finding in the spermatogenesis of M. glacialis is that the formation of the pro-acrosomal vesicles seems to be initiated in late spermatogonia. Small dense bodies resulting from the division of large granulofibrillar masses were also observed in the cytoplasm of late spermatogonia. During spermiogenesis the inner acrosomal vesicle membrane becomes coated first with dense materials originated from the cytoplasmic dense bodies and then with cisternae of endoplasmic reticulum. Both coating materials are incorporated in the periacrosomal space of the mature acrosome. Besides being involved in the genesis of the periacrosomal material, cytoplasmic dense bodies were also seen in close relationship with intercellular bridges and midpiece structures of spermatids. These findings are discussed in comparison with other echinoderm spermatogenesis.