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Neuropeptides are one of the largest and most diverse families of signaling molecules in animals and, accordingly, they regulate many physiological processes and behaviors. Genome and transcriptome sequencing has enabled the identification of genes encoding neuropeptide precursor proteins in species from a growing variety of taxa, including bilaterian and non-bilaterian animals. Of particular interest are deuterostome invertebrates such as the phylum Echinodermata, which occupies a phylogenetic position that has facilitated reconstruction of the evolution of neuropeptide signaling systems in Bilateria. However, our knowledge of neuropeptide signaling in echinoderms is largely based on bioinformatic and experimental analysis of eleutherozoans—Asterozoa (starfish and brittle stars) and Echinozoa (sea urchins and sea cucumbers). Little is known about neuropeptide signaling in crinoids (feather stars and sea lilies), which are a sister clade to the Eleutherozoa. Therefore, we have analyzed transcriptome/genome sequence data from three feather star species, Anneissia japonica, Antedon mediterranea, and Florometra serratissima, to produce the first comprehensive identification of neuropeptide precursors in crinoids. These include representatives of bilaterian neuropeptide precursor families and several predicted crinoid neuropeptide precursors. Using A. mediterranea as an experimental model, we have investigated the expression of selected neuropeptides in larvae (doliolaria), post-metamorphic pentacrinoids and adults, providing new insights into the cellular architecture of crinoid nervous systems. Thus, using mRNA in situ hybridization F-type SALMFamide precursor transcripts were revealed in a previously undescribed population of peptidergic cells located dorso-laterally in doliolaria. Furthermore, using immunohistochemistry a calcitonin-type neuropeptide was revealed in the aboral nerve center, circumoral nerve ring and oral tube feet in pentacrinoids and in the ectoneural and entoneural compartments of the nervous system in adults. Moreover, functional analysis of a vasopressin/oxytocin-type neuropeptide (crinotocin), which is expressed in the brachial nerve of the arms in A. mediterranea, revealed that this peptide causes a dose-dependent change in the mechanical behavior of arm preparations in vitro—the first reported biological action of a neuropeptide in a crinoid. In conclusion, our findings provide new perspectives on neuropeptide signaling in echinoderms and the foundations for further exploration of neuropeptide expression/function in crinoids as a sister clade to eleutherozoan echinoderms.

The mutable collagenous tissue (MCT) of echinoderms has the capacity to undergo changes in its tensile properties within a timescale of seconds under the control of the nervous system. All echinoderm autotomy (defensive self-detachment) mechanisms depend on the extreme destabilisation of mutable collagenous structures at the plane of separation. This review illustrates the role of MCT in autotomy by bringing together previously published and new information on the basal arm autotomy plane of the starfish Asterias rubens L. It focuses on the MCT components of breakage zones in the dorsolateral and ambulacral regions of the body wall, and details data on their structural organisation and physiology. Information is also provided on the extrinsic stomach retractor apparatus whose involvement in autotomy has not been previously recognised. We show that the arm autotomy plane of A. rubens is a tractable model system for addressing outstanding problems in MCT biology. It is amenable to in vitro pharmacological investigations using isolated preparations and provides an opportunity for the application of comparative proteomic analysis and other “-omics” methods which are aimed at the molecular profiling of different mechanical states and characterising effector cell functions.

The mutable collagenous tissue (MCT) of echinoderms (starfish, sea-urchins and their close relations) has the capacity to drastically alter its mechanical properties within a timescale of seconds under the control of the nervous system. MCT is of central importance to the biology of echinoderms: postural fixation of the body or its appendages depends on passive MCT stiffening, and all autotomy (defensive self-detachment) mechanisms involve irreversible MCT destabilisation. This chapter reviews research on the biology and biomedical applications of MCT. It focuses on recent advances in knowledge of its organisation, biomechanics, molecular mechanisms of tensile change and neural control and demonstrates that MCT has attracted interest as a source of constituents for the construction of novel materials with potential biomedical applications and as a source of inspiration for the design of entirely artificial materials with adaptable and controllable mechanical properties.

Marine biodiversity is expressed through the huge variety of vertebrate and invertebrate species inhabiting intertidal to deep-sea environments. The extraordinary variety of â forms and functionsâ  exhibited by marine animals suggests they are a promising source of bioactive molecules and provides potential inspiration for different biomimetic approaches. This diversity is familiar to biologists and has led to intensive investigation of metabolites, polysaccharides, and other compounds. However, marine collagens are less well-known. This review will provide detailed insight into the diversity of collagens present in marine species in terms of their genetics, structure, properties, and physiology. In the last part of the review the focus will be on the most common marine collagen sources and on the latest advances in the development of innovative materials exploiting, or inspired by, marine collagens., The authors are grateful for the financial support from European Union, under the scope of European Regional Development Fund((ERDF) through the POCTEP project 0687_NOVOMAR_1_P and Structured Project NORTE-01- 0145-FEDER-000021 and from the Portuguese Foundation for Science and Technology (FCT), under the scope of the BiogenInk project (M-ERA-NET2/0022/2016) and from the European Cooperation in Science & Technology program (EU COST). Grant title: “Stem cells of marine/aquatic inverte brates: from basic research to innovative applications” (MARISTEM). MSR acknowledges FCT for the Ph.D. scholarship (PD/BD/143091/2018).

The dental apparatus of cidarid echinoids represents one of the two fundamental types of lanterns evolved in the sea-urchins and represents an ideal model to apply the multiple approach of study already proposed for the well-known camarodont type. The lantern of Stylocidaris consists of the usual differently interacting components which display a number of peculiar features from both a macro- and a microstructural point of view. Its remarkable structural adaptations can be related to its peculiar functional properties, their full significance being revealed only when analysed in the context of the integrated biomechanical model.

The peristomial plates are skeletal components of the interbrachial frame (or mouth frame), which is located below the true mouth of ophiuroids. Whilst the peristomial plates were extensively described and used as diagnostic characters by some early workers, for the past 100 years they have been largely neglected as a taxonomic resource. In this investigation the peristomial plates of 48 species representing 21 families were examined directly, and information on a further 61 species, including representatives of another eight families, was obtained from the published literature. Observations were made with regard to fragmentation state, relative size and orientation of the peristomial plates. Although fragmentation state showed little consistency at any taxonomic level, relative size and orientation segregated a group of families comprising species with relatively small, inclined peristomial plates, viz. Ophiotrichidae, Ophiopholidae, Ophiactidae, Amphiuridae and Ophiocomidae, together with a single hemieuryalid species ? Ophioplocus januarii. The distribution of peristomial plate traits was strongly correlated with that of several other character states pertaining to the interbrachial frame. This supported the proposition that two major types of interbrachial frame are present in ophiuroids (designated ?A? and ?B?). Current phylogenies inferred from both morphological and molecular data imply that type B is derived and has evolved independently at least twice in the orders Amphilepidida and Ophiacanthida. This represents a remarkable example of evolutionary convergence. An analysis of the distribution of all interbrachial frame character states suggested that within the Amphilepidida paedomorphosis was probably responsible for the complete reversion of the interbrachial frame to the ancestral type A condition in two families (Ophiothamnidae and Amphilepididae) of suborder Gnathophiurina and possibly responsible for varying degrees of trait reversal in the four families of suborder Ophionereidina. Such paedomorphic events may have been associated with a secondary return to the deep-sea from shallow-sea environments. Fil: Wilkie, Iain C.. University of Glasgow; Reino Unido Fil: Brogger, Martin Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes within physiological timescales, the only major exception being the reversible destiffening of the mammalian uterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (sea urchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions in timescales of around a second to minutes. Elucidation of the molecular mechanism underlying such mutability has implications for the zoological, ecological and evolutionary field. Important information could also arise for veterinary and biomedical sciences, particularly regarding the pathological plasticization or stiffening of connective tissue structures. In the present investigation we analyzed aspects of the ultrastructure and biochemistry in two representative models, the compass depressor ligament and the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three different mechanical states. The results provide further evidence that the mechanical adaptability of echinoderm connective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher glycosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compass depressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues. The possible involvement of GAG in the mutability phenomenon will need further clarification. During the shift from a compliant to a standard condition, significant changes in GAG content were detected only in the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling) and biochemistry (two alpha chains) were found between the two models and mammalian collagen. Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLAST alignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen.

The mechanically adaptable connective tissue of echinoderms (Mutable Collagenous Tissue—MCT), which can undergo drastic nervously-mediated changes in mechanical properties, represents a promising model for biomaterial design and biomedical applications. MCT could be a source of, or an inspiration for, new composite materials whose molecular inter­actions and structural conformation can be changed in response to external stimuli. MCT is composed mostly of collagen fibrils, comparable to those of mammals, plus a variety of other components, including other fibrillar structures, proteo­glycans and glycoproteins. This contribution presents the preliminary results of a detailed analysis of MCT components in the sea-urchin Paracentrotus lividus, focusing on biochemical characterization of the fibrils and biomolecular analysis of the presumptive glycoproteins involved. The final aims will be to confirm the presence and the role of these glycoproteins in echinoids and to manipulate simpler components in order to produce a composite with mutable mechanical properties.

Chondrosia reniformis is a common marine demosponge that shows striking tissue plasticity and unusual body deformability. This sponge can develop long and slender outgrowths extending from the parental body. According to some authors, this phenomenon, called “creeping”, can be related to asexual reproduction, atypical mechanisms of «localized» locomotion or passive response to environmental stress. Here we address this phenomenon by means of an interdisciplinary approach consisting of field survey, experimental field studies and experimental laboratory studies. During field survey and field experimental survey we observed that the instability of substratum is an important factor that trigs the beginning of creeping. The sponge size does not seem to be directly involved in the occurrence of the phenomenon. Specimens of Bergeggi (Ligurian Sea, northern Italy) show a high correlation between the creeping phenomenon and the sea temperature; this seems to support the hypothesis that the phenomenon is related to asexual reproduction, which is in its turn seasonally regulated by environmental temperature. In addition, experimental laboratory studies performed in different mechanical conditions on isolated samples of both ectosome and choanosome showed that temperature affects mesohyl mechanical properties: the lower is the temperature the stiffer is the mesohyl. The different physiological states recorded by the laboratory experiments are expressions of the mechanical adaptability of the collagenous mesohyl of C. reniformis and suggest that stiffness variability is under cellular control. On the basis of present results we can infer that C. reniformis can exert some control on the creeping phenomenon and that the primary factors implied in inducing creeping phenomena are the instability of substratum and the temperature. Interestingly the capability to modulate the mechanical properties of the collagenous matrix is an uncommon feature that C. reniformis shares with the mutable collagenous tissue (MCT) of Echinoderms. This close analogy, which is supported by morphological and physiological evidence, is an intriguing point that opens a wide range of evolutionary and functional questions.

Echinoderms possess unique connective tissues, called mutable collagenous tissues (MCTs), which undergo nervously mediated, drastic and reversible or irreversible changes in their mechanical properties. Connective tissue mutability influences all aspects of echinoderm biology and is a key-factor in the ecological success of the phylum. Due to their sensitivity to endogenous or exogenous agents, MCTs may be targets for a number of common pollutants, with potentially drastic effects on vital functions. Besides its ecological relevance, MCT represents a topic with relevance to several applied fields. A promising research route looks at MCTs as a source of inspiration for the development of novel biomaterials. This contribution presents a review of MCT biology, which incorporates recent ultrastructural, biomolecular and biochemical analyses carried out in a biotechnological context.

As well as acting as a neurotransmitter, serotonin is also involved in morphogenetic signalling during crucial phases of many developmental and regenerative processes such as cleavage, migration and differentiation. Echinoderms display nerve-dependent regenerative phenomena and serotonin is one of the main neural regulatory factors that have been identified in them. The present work was designed to investigate the broad-spectrum involvement of this molecule in echinoderm regeneration, focusing on arm regeneration in the crinoidAntedon mediterranea. We carried out specificin vivoexposure experiments to selected anti-serotonergic drugs (a synthesis inhibitor: parachlorophenylalanine and a receptor antagonist: methiothepin) and explored their possible effects on arm regeneration. Drug exposure appeared to affect regeneration, causing a general delay of regenerative growth and several histological anomalies mainly in the muscles of the stump. In addition, exposure to the antagonist produced a marked reduction of cell proliferation in both the regenerate and the stump. Our results provide further evidence that serotonin has wider functions than those related to interneuronal communication and that it may be a critical signalling molecule in the main processes of regeneration such as proliferation, morphogenesis and differentiation.

The ophiuroid Amphipholis kochii is able to detach its central disc from the underlying oral frame in response to external stimuli. In this article we supply new observations on the microanatomy and ultrastructure of the autotomy plane, and of the juxtaligamental system which is believed to bring about connective tissue changes that underpin the detachment process. We correct previous confusion over the innervation of juxtaligamental nodes involved in disc autotomy, provide evidence that juxtaligamental cells are a population of specialized nerve cells, and present observations on changes in the ultrastructure of juxtaligamental cells during autotomy, which support the view that they are responsible for connective tissue disruption.

We found that ligaments connecting the skeletal elements in the arm of the brittlestar Amphipholis kochii, consisted of mutable collagenous tissue (MCT), whose basichistological character is that of numerous processes of juxtaligamental cells penetrating the extracellular matrix. These cells are located in the hyponeural neuroepithelium associated with hyponeural nerves, and were also recorded in the spine ganglia and basiepithelial nervous plexus in the coelomic epithelium. The distinctive feature of juxtaligamental cells is the presence of electron-dense granules in their cytoplasm and a well developed synthetic apparatus. Based on personal and literature data it is concluded that juxtaligamental cells are a part of the nervous system, and their basic function is related to providing MCT with innervation.

The compass depressors (CDs) of the sea-urchin lantern are ligaments consisting mainly of discontinuous collagen fibrils associated with a small population of myocytes. They are mutable collagenous structures, which can change their mechanical properties rapidly and reversibly under nervous control. The aims of this investigation were to characterise the baseline (i.e. unmanipulated) static mechanical properties of the CDs of Paracentrotus lividus by means of creep tests and incremental force-extension tests, and to determine the effects on their mechanical behaviour of a range of agents. Under constant load the CDs exhibited a three-phase creep curve, the mean coefficient of viscosity being 561±365 MPa.s. The stress-strain curve showed toe, linear and yield regions; the mean strain at the toe-linear inflection was 0.86±0.61; the mean Young's modulus was 18.62±10.30 MPa; and the mean tensile strength was 8.14±5.73 MPa. Hyaluronidase from Streptomyces hyalurolyticus had no effect on creep behaviour, whilst chondroitinase ABC prolonged primary creep but had no effect on secondary creep or on any force-extension parameters; it thus appears that neither hyaluronic acid nor sulphated glycosaminoglycans have an interfibrillar load transfer function in the CD. Acetylcholine, the muscarinic agonists arecoline and methacholine, and the nicotinic agonists nicotine and 1-[1-(3,4-dimethyl-phenyl)-ethyl]-piperazine produced an abrupt increase in CD viscosity; the CDs were not differentially sensitive to muscarinic or nicotinic agonists. CDs showed either no, or no consistent, response to adrenaline, L-glutamic acid, 5-hydroxytryptamine and γ-aminobutyric acid. Synthetic echinoid tensilin-like protein had a weak and inconsistent stiffening effect, indicating that, in contrast to holothurian tensilins, the echinoid molecule may not be involved in the regulation of collagenous tissue tensility. We compare in detail the mechanical behaviour of the CD with that of mammalian tendon and highlight its potential as a model system for investigating poorly understood aspects of the ontogeny and phylogeny of vertebrate collagenous tissues., (undefined), info:eu-repo/semantics/publishedVersion

SUMMARYThe mutable collagenous tissue (MCT) of echinoderms has the capacity to change its mechanical properties in a time scale of less than 1 s to a few minutes under the influence of the nervous system. Although accumulating evidence indicates that the mechanical adaptability of MCT is due primarily to the modulation of interactions between components of the extracellular matrix, the presence of muscle in a few mutable collagenous structures has led some workers to suggest that contractile cells may play an important role in the phenomenon of variable tensility and to call for a re-evaluation of the whole MCT concept. This contribution summarises present information on MCT and appraises the argument implicating muscle in its unique mechanical behaviour. It is concluded that there is no evidence that the variability of the passive mechanical properties of any mutable collagenous structure is due to muscle.

Although sponges are still often considered to be simple, inactive animals, both larvae and adults of different species show clear coordination phenomena triggered by extrinsic and intrinsic stimuli. Chondrosia reniformis, a common Mediterranean demosponge, lacks both endogenous siliceous spicules and reinforcing spongin fibers and has a very conspicuous collagenous mesohyl. Although this species can stiffen its body in response to mechanical stimulation when handled, almost no quantitative data are available in the literature on this phenomenon. The present work was intended to quantify the dynamic response to mechanical stimulation both of intact animals and isolated tissue samples in order to evaluate: (i) the magnitude of stiffening; (ii) the relationship between the amount of stimulation and the magnitude of the stiffening response; (iii) the ability of the whole body to react to localized stimulation; (iv) the possible occurrence of a conduction mechanism and the role of the exopinacoderm (outer epithelium). Data on mesohyl tensility obtained with mechanical tests confirmed the difference between stimulated and non-stimulated isolated tissue samples, showing a significant relationship between ectosome stiffness and the amount of mechanical stimulation. Our experiments revealed a significant difference in tensility between undisturbed and maximally stiffened sponges and evidence of signal transmission that requires a continuous exopinacoderm. We also provide further evidence for the presence of a chemical factor that alters the interaction between collagen fibrils, thereby changing the mechanical properties of the mesohyl.

The exterior coelomic septum (ECS) is a mesentery-like structure that encloses the lantern of regular sea-urchins and connects it to the inner surface of the test. This paper describes the ultrastructure and microarchitecture of the ECS in Stylocidaris affinis (Cidaridae, Echinoida) and provides information on its contractile and passive mechanical properties. The ECS forms five interambulacral pouches each of which has adthecal (test-facing) and adambulacral (ambulacrum-facing) walls. The ECS wall comprises two coelothelia separated by a layer of connective tissue. The outer coelothelium is a single layer of monociliated cuboidal peritoneocytes and basally located axon-like processes. The inner coelothelium is a single layer of squamous peritoneocytes overlying axon-like processes and, in the adthecal regions only, parallel arrays of elongated myocytes orientated obliquely or horizontally. The intraseptal connective tissue consists mainly of collagen fibrils with sparsely distributed spherule cells and cells containing heterogeneous vesicles. In the adambulacral regions of the ECS hollow beaded microfibrils 20–23 nm in diameter form fibre-like aggregations. This layer also contains calcite spicules of variable size, shape, abundance and orientation. Isolated preparations of the ECS show concentration-dependent contractile responses to K+ ions and acetylcholine. The magnitude of the contractile force varies with the vertical position of the lantern (which determines the starting length of the ECS) in an unusual pattern. Cyclical loading-unloading tests indicate that, as the lantern is raised, the ECS shows low stiffness until the lantern reaches its normal resting position. It is concluded that the adthecal regions of the ECS help to set a limit to lantern retraction and that their contractility assists the protractor muscles in exerting a downward pull on the lantern.

Certain components of the jaw apparatus, or lantern, of regular sea-urchins form a ’compass system’ the function of which has still to be established. This system includes ten compass depressors (CDs) which connect the compass ossicles to the inner edge of the test. Previous studies focused on the CDs of euechinoid sea-urchins. This paper provides the first detailed account of the organization and mechanical behaviour of the CDs of a cidaroid sea-urchin, Stylocidaris affinis. The outermost layer of its CDs is a coelothelium comprising apical peritoneocytes, sparse subapical myocytes and two types of granule-containing cells. The central core of the CD is dominated by longitudinally orientated collagen fibrils arranged in bundles surrounded by networks of beaded microfibrils. Myocytes are scattered throughout the core and are always surrounded by a basal lamina which usually encloses the same two types of granule-containing cells observed in the coelothelium. Each CD is attached to the outer coelomic septum by a mesentery, both the connective tissue and coelothelia of which lack myocytes and granule-containing cells. From data on the relationship between the vertical position of the lantern and the passive and active forces developed by intact CD sets, it appears that the CDs resist elevation of the lantern above its ’resting position’ and that they develop a maximal contractile force when their length corresponds to that at the resting position. The connective tissue of the CDs has the capacity to undergo nervously mediated changes in stretch resistance, although this is expressed more weakly than in the euechinoid CDs. These results are related to current views on the physiological role of the compass system.

The jaw apparatus, or lantern, of sea-urchins contains five pairs of retractor and protractor muscles which are responsible for lantern displacement. Using intact retractor or protractor groups, the force-length relations of these muscles were compared in two taxonomically distant species, Paracentrotus lividus and Stylocidaris affinis. The total contractile forces generated by the muscles can be resolved into vertical and horizontal components. It was found that the vertical component of the retractors is maximal at a lantern position which is significantly lower (i.e. more protruded) in Paracentrotus than in Stylocidaris. Total forces generated by the retractors were in both species maximal at or above the lantern `resting positions'. In Paracentrotus alone, the total force-displacement curves tended to be bimodal. It is hypothesized that the retractors of Paracentrotus contain two populations of muscle fibres, one adapted for jaw opening and one for lantern retraction. No significant differences in the properties of the protractors of the two species could be identified. The lantern of Paracentrotus is more mobile than that of Stylocidaris and is able to exploit a wider range of food sources. This investigation has shown that the force-length relations of the lantern muscles match their differing working conditions.

Echinoderms and sponges share a unique feature that helps them face predators and other environmental pressures. They both possess collagenous tissues with adaptable viscoelastic properties. In terms of morphology these structures are typical connective tissues containing collagen fibrils, fibroblast- and fibroclast-like cells, as well as unusual components such as, in echinoderms, neurosecretory-like cells that receive motor innervation. The mechanisms underpinning the adaptability of these tissues are not completely understood. Biomechanical changes can lead to an abrupt increase in stiffness (increasing protection against predation) or to the detachment of body parts (in response to a predator or to adverse environmental conditions) that are regenerated. Apart from these advantages, the responsiveness of echinoderm and sponge collagenous tissues to ionic composition and temperature makes them potentially vulnerable to global environmental changes.

The peristomial membrane (PM) is the area of flexible body wall which surrounds the mouth of regular sea-urchins. In cidaroid sea-urchins, like Stylocidaris. affnis (Phil.), it contains an extensive endoskeleton of overlapping plates. This paper describes the basic mechanical properties of the PM of S. affinis and compares them with those of the PM of the previously studied echinacean euechinoid Paracentrotus lividus. When subjected to vertical deformation, the PM of S. affinis generates J-shaped tension-deformation curves with an inflexion point at a deformation corresponding to about 3% of the perignathic girdle diameter. It is considerably stiffer than the P. lividus PM and, unlike the latter, it shows asymmetrical properties, i.e. greater resistance to aboral flexion (retraction) than to oral flexion (protraction). In interambulacral regions the junction between the PM endoskeleton and test of S. affinis takes the form of a wide ligament-filled gap which acts as a compliant hinge. In marked contrast to the echinacean PM, that of S. affinis provides little evidence for the presence of mutable collagenous tissue (MCT). The cidaroid PM is interpreted as a design which maximizes mechanical defence whilst retaining the capacity for some flexibility. A possible correlation between the lantern systems and feeding habits of sea-urchins is discussed.

The skeletal morphology of the arm spine joint of the brittlestar Ophiocomina nigra was examined by scanning electron microscopy and the associated epidermis, connective tissue structures, juxtaligamental system and muscle by optical and transmission electron microscopy. The behaviour of spines in living animals was observed and two experiments were conducted to establish if the spine ligament is mutable collagenous tissue: these determined (1) if animals could detach spines to which plastic tags had been attached and (2) if the extension under constant load of isolated joint preparations was affected by high potassium stimulation. The articulation normally operates as a flexible joint in which the articular surfaces are separated by compliant connective tissue. The articular surfaces comprise a reniform apposition and peg-in-socket mechanical stop, and function primarily to stabilise spines in the erect position. Erect spines can be completely immobilised, which depends on the ligament having mutable tensile properties, as was inferred from the ability of animals to detach tagged spines and the responsiveness of isolated joint preparations to high potassium. The epidermis surrounding the joint has circumferential constrictions that facilitate compression folding and unfolding when the spine is inclined. The interarticular connective tissue is an acellular meshwork of collagen fibril bundles and may serve to reduce frictional forces between the articular surfaces. The ligament consists of parallel bundles of collagen fibrils and 7-14 nm microfibrils. Its passive elastic recoil contributes to the re-erection of inclined spines. The ligament is permeated by cell processes containing large dense-core vesicles, which belong to two types of juxtaligamental cells, one of which is probably peptidergic. The spine muscle consists of obliquely striated myocytes that are linked to the skeleton by extensions of their basement membranes. Muscle contraction may serve mainly to complete the process of spine erection by ensuring close contact between the articular surfaces.

The functional organization of both the epidermis and the coelomic lining of the peristomial membrane in the regular sea‐urchin Paracentrotus lividus is described and correlated with the extreme deformability of the peristomial membrane and with the ≪mutability≫ of its related dermis. The epidermis consists of a monolayer of columnar supporting cells mixed with ciliated sensory cells, the wide interspaces between these epithelial cells being occupied apically by a variety of cell types and basally by a conspicuous basiepithelial nerve plexus. The coelomic lining is a peripharyngeal coelomic epithelium consisting of apical monociliated peritoneocytes and subapical myocytes, and has an associated basiepithelial nerve plexus. Although the basic morphology of these epithelia is similar to that of corresponding tissues in other echinoderms, they both have significant structural adaptations and display the histological pattern of typical transitional epithelia, which enables them to match the wide rang...

Although l-glutamate is the most widespread excitatory neurotransmitter in vertebrate and invertebrate nervous systems, there is only sparse evidence that it has this role in echinoderms. Following our previous finding that l-glutamate is widely distributed in the arms of the featherstar (crinoid echinoderm) Antedon mediterranea and initiates arm autotomy (defensive detachment), we now provide evidence of glutamatergic involvement in the control of the arm muscles of the same species using immunocytochemical and physiological methods. Immunofluorescence and immunoenzymatic techniques, which employed the same polyclonal antibody against l-glutamate conjugated to glutaraldehyde, revealed a high level of glutamate-like reactivity in the brachial muscles. By recording the mechanical responses of isolated arm pieces, we found that l-glutamate, l-aspartate and elevated [K(+)](o) induced rhythmic muscle contractions, while glycine, γ-aminobutyric acid, adrenaline and acetylcholine had either no, or no consistent, effect. The frequency and duration of the dominant component of the rhythmic contractions indicated that these may be responsible for the rhythmic activity of the arms that occurs during swimming and after autotomy. We conclude that it is highly likely that l-glutamate has at least a neuromodulatory role in the neural pathways controlling the brachial muscles of A. mediterranea.

The stalk of isocrinid sea-lilies consists largely of skeletal plates linked by collagenous ligaments. Although lacking contractile tissue, it can bend in response to external stimuli. The stalk of Cenocrinus asterius was tested mechanically to determine whether the mechanical properties of its ligaments are under physiological control. In bending tests, ligaments at the mobile symplexal junctions showed a limited “slackening” response to high K + concentrations which was blocked reversibly by the anaesthetic propylene phenoxetol. In bending tests and uniaxial loading tests, ligaments at the normally rigid synostosal junctions ruptured in response to high K + , confirming that these junctions are specialized for autotomy. It is concluded that the ligaments are mutable collagenous structures whose presence explains the mechanical versatility of the isocrinid stalk.

The peristomial membrane is an area of flexible body wall which connects the test to the lantern or masticatory apparatus of regular sea‐urchins. The peristomial membrane of Paracentrotus lividus was examined by various light microscopical techniques and by scanning and transmission electron microscopy, and its mechanical properties were investigated by load‐deformation tests. Attention was focused on the collagenous dermis which consists mainly of crossed‐fibre arrays arranged in three histologically distinct sublayers. This microarchitectural arrangement gives rise to nonlinear load‐deformation characteristics: when subjected to vertical deformation such as would occur during lantern protraction, the peristomial membrane shows firstly low stiffness and then high stiffness phases. The response of the peristomial membrane to chemical agents suggests that the tensile properties of the dermis are under physiological control. Our results indicate that the collagenous fibres of the dermis belong to s...

In the present analysis experimental results and computer modelling have been used to compare biomechanics of the two most contrasting lantern types among regular echinoids, i.e. the cidarid-type (Stylocidaris affinis) and the camarodont (Paracentrotus lividus) lantern. The lantern is modelled as a rigid pyramid anchored in the centre of the peristomial area. Each side is connected to the test by means of muscles (protractors and retractors) rind ligaments (compass depressors). The overall movement of the lantern may be resolved into vertical displacement and lateral tilting. Computer-aided mechanical analysis of the system takes into account the action of protractor and/or retractor muscles, their elastic reaction and the interaction with the ligamentous structures (peristomial membrane and compass depressors). The geometrical parameters have been determined by preliminary structural analysis and the values of active and passive forces have been measured experimentally. The results of simulations highlight important differences in the mechanics of the Stylocidaris lantern compared to that of Paracentrotus: (1) a very much greater antagonistic effect of muscular passive stretch resistance on lateral tilting: (2) a minor role of the peristomial membrane tensile strength and muscular activity in controlling vertical lantern displacement: (3) a less efficient lantern retraction and protraction owing to unfavourable muscle insertion position on the perignathic girdle and ineffective muscular contraction forces. As a conclusion, in Stylocidaris the action of the protractor and retractor muscles seems to play no significant role in moving the overall lantern, their action being mainly exerted to stabilize it. On the contrary, in Paracentrotus such stabilizing action is provided by the compass depressor ligaments. Finally, on theoretical grounds the arrangement of muscle attachments around the perignathic girdle in Stylocidaris seems to be just as good as that of Paracentrotus for controlling overall lantern mobility, if relative heights of insertions are ignored.

The Aristotle's lantern, or masticatory apparatus, of regular sea-urchins is a complex musculo-skeletal system which is thought to have contributed significantly to the evolutionary success of these animals. This paper gives an account of the antomical relationships and functional morphology of both skeletal and soft tissue components in the lantern and related structures of the sea-urchin Stylocidaris affinis (Cidaridae), and compares these features with their equivalent in the previously described lantern of the sea-urchin Paracentrotus lividus (Echinidae, Camarodonta). There are major differences in the skeletons of these lanterns which involve mostly the arrangement and morphology of elements participating in movement, i.e. joints and articular surfaces, and which highlight the generally heavier and less mobile nature of the lantern in the Cidaridae. There are remarkably few differences, however, in the microstructure of the skeletal stereom. Significant dissimilarities were found in the anatomical arrangement of muscles and ligamentous structures and in their macro- and microstructure. The implications of these morphological features for the functioning of the lantern of the Cidaridae are discussed in the context of an integrated model of lantern biomechanics.

SUMMARYThe crinoid echinoderm Antedon mediterranea autotomises its arms at specialised skeletal joints known as syzygies that occur at regular intervals along the length of each arm. Detachment is achieved through the nervously mediated destabilisation of ligament fibres at a particular syzygy. The aim of this investigation was to identify neurotransmitters that are involved in the autotomy response. Physiological experiments were conducted on isolated preparations of syzygial joints, which can be induced to undergo autotomy-like fracture by applying stimulatory agents such as elevated [K+]o. Initial experiments with elevated [K+]o showed that the autotomy threshold (the minimum amount of stimulation required to provoke autotomy) is lowest in syzygies at the arm base and rises distally. Of a range of neurotransmitter agonists tested, only l-glutamate invoked syzygial destabilisation, as did its analogues l-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, but not l-(+)-2-amino-4-phosphonobutyrate (l-AP4) or N-methyl-d-aspartate (NMDA). The implication that l-glutamate stimulates syzygial fracture through AMPA/kainate-like receptors was supported by the finding that the action of l-glutamate was inhibited by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Acetylcholine depressed the response of syzygial preparations to l-glutamate, suggesting a possible mechanism by which the autotomy threshold could be varied constitutively and facultatively. An immunocytochemical method employing a polyclonal antibody against l-glutamate conjugated to glutaraldehyde revealed l-glutamate-like immunoreactivity in all components of the putative neural pathway controlling the autotomy reflex, including the epidermis, brachial nerve, syzygial nerves and cellular elements close to the syzygial ligaments. We conclude that it is highly probable that l-glutamate acts as an excitatory neurotransmitter in the activation of arm autotomy in A. mediterranea.

The compass depressors are bands of soft tissue which connect the compass ossicles of the echinoid lantern to the inner edge of the test. They are essentially ligaments with on one side a thin layer of muscle cells. The ligamentous component consists mainly of a parallel array of collagen fibrils with interspersed 12 nm microfibrils. The most notable cellular constituents are granule-containing cell bodies and their processes which resemble the juxtaligamental cells that have been found in all echinoderm mutable collagenous tissues and which may control the tensility of these tissues. The muscle cells occupy about 8% of the total cross-sectional area of the compass depressor and are located in a richly innervated pseudostratified myoepithelium. When subjected to constant low loads in creep tests the compass depressor stretches to a fixed length beyond which there is no further extension. The length at this creep limit coincides with the maximum length to which the compass depressor is stretched by natural movements of the intact lantern. Stress-strain tests show that treatment with 1 mM acetylcholine or 100 mM K+ ions can increase reversibly the stiffness of the compass depressor to an extent that cannot be due to contraction of the myoepithelium, suggesting that the mechanical properties of the ligament are under physiological control. Tension-length data on the myoepithelium suggest that it generates a maximum active tension when the compass depressor is stretched to the creep limit. The implications of these results for the function of the compass depressors are discussed.

SUMMARY The marine sponge Chondrosia reniformis Nardo consists largely of a collagenous tissue, the mesohyl, which confers a cartilaginous consistency on the whole animal. This investigation was prompted by the incidental observation that, despite a paucity of potentially contractile elements in the mesohyl, intact C. reniformis stiffen noticeably when touched. By measuring the deflection under gravity of beam-shaped tissue samples, it was demonstrated that the flexural stiffness of the mesohyl is altered by treatments that influence cellular activities, including [Ca2+]manipulation, inorganic and organic calcium channel-blockers and cell membrane disrupters, and that it is also sensitive to extracts of C. reniformis tissue that have been repeatedly frozen then thawed. Since the membrane disrupters and tissue extracts cause marked stiffening of mesohyl samples, it is hypothesised that cells in the mesohyl store a stiffening factor and that the physiologically controlled release of this factor is responsible for the touch-induced stiffening of intact animals.

This paper provides the first detailed account of the histological and ultrastructural organisation of an echinoderm ligament that is non-mutable. Each of the five compass ossicles on the aboral side of the lantern of regular echinoids is linked to an underlying rotular ossicle by a compass-rotular ligament (CRL). The structure and anatomical relations of the CRL of Echinus esculentus L. were examined by light microscopy and scanning and transmission electron microscopy, and its responsiveness to neuroactive agents was observed in stress relaxation tests. The CRL consists of (1) thick collagen fibres that form an outer crossed fibre lattice and an internal system of parallel suspensory fibres and (2) regions composed mainly of bundles of ca. 12 nm microfibrils. The CRL is sparsely cellular, most cell bodies belonging to heterogeneous vesicle-containing cells that undergo apoptosis-like cytoplasmic fragmentation and produce linear aggregations of cell fragments. Coelothelia investing the CRL show intense secretory activity reminiscent of lamellar body production by mammalian mesothelia and are the source of bundles of cell processes that penetrate deeply into the CRL, branch sparingly and terminate between the thick fibres. Isolated CRL preparations are unresponsive to seawater containing 1 mM acetylcholine or 100 mM K+, but show a transient increase in stress relaxation rate when treated with 0.56 M KCl. The effects of solutions of other alkali metal chlorides suggest that the latter response is due to the direct action of KCl on extracellular components of the CRL and is not nervously mediated.

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states (“standard”, “compliant” and “stiff”) was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability.

The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs.

The biology of asexual reproduction in the fissiparous brittle star Ophiocomella ophiactoides (H.L. Clark) at Jamaica, W.I. was studied during June, July, August and December, 1981 and July, 1982. Most O. ophiactoides were in various stages of recovery following fission, having three long and three shorter arms. Recently split specimens with only three arms and half of a disc were present in winter and summer suggesting that asexual reproduction occurs throughout the year. A small number of five- and seven-armed individuals resulted from natural variability in fission and regeneration processes. Fission in O. ophiactoides proceeded unilaterally, with one side of the disc first apparently softening to form a furrow which then progressed across the disc to separate the brittle star into two halves. Initiation of fission appeared to be internally controlled. Fission occurred before the new arms were fully regenerated. This minimized the interval between successive fissions and so maximized increase in numbers due to asexual reproduction. The fission plane was not predetermined but variable which ensured that many halves acquire a mixture of both longer and shorter arms. It was deduced from data on arm configuration and regeneration rates that the interval between fissions was of the order of 89 days. A single specimen potentially may divide and produce 15 new individuals each year. The population size structure showed that larval recruitment was absent or minimal. Populations of O. ophiactoides at Jamaica must be maintained by asexual reproduction.

Mutable collagenous tissues (MCTs) of echinoderms can be regarded as intelligent and dynamic biomaterials, due to their ability to reversibly change their mechanical properties in a short physiological time span. This mutability phenomenon is nervously mediated and involves secreted factors of the specialized 'juxtaligamental' cells, which, when released into the extracellular matrix (ECM), change the cohesive forces between collagen fibrils. MCTs exist in nature in several forms, including some associated with echinoderm autotomy mechanisms. Since the molecular mechanism of mutability is still incompletely understood, the aim of this work was to provide a detailed biochemical analysis of a typical mutable collagenous structure and to identify possible correlations between its biochemistry and mechanical states. A better understanding of the mutability phenomena is likely to provide a unique opportunity to develop new concepts that can be applied in the design of dynamic biomaterial for tissue regeneration, leading to new strategies in regenerative medicine. The MCT model used was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was analyzed in different mechanical states, mimicking the mutability phenomenon. Spectroscopic techniques, namely Fourier transform infrared (FT-IR) and confocal Raman microscopy, were used to identify the specific molecular components that contribute to the CDL biochemical microenvironment and to investigate the possibility that remodelling/synthesis of new ECM components occurs during the mutability phenomenon by analogy with events during pregnancy in the uterine cervix of mammals (which also consists mainly of mechanically adaptable connective tissues). The results demonstrate that CDL ECM includes collagen with biochemical similarities to mammalian type I collagen, as well as sulphated glycosaminoglycans (GAGs). CDL mutability seems to involve a molecular rearrangement of the ECM, without synthesis of new ECM components. Although there were no significant biochemical differences between CDLs in the various mechanical states were observed. However, subtle adjustments in tissue hydration seemed to occur, particularly during stiffening.