[Show abstract][Hide abstract] ABSTRACT: Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30–400 nm and mesh < 2 μm) and
Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy; E-Mails: email@example.com (C.D.B.); firstname.lastname@example.org (V.A.); email@example.com (D.F.); firstname.lastname@example.org (E.C.); email@example.com (F.B.); firstname.lastname@example.org (M.D.C.C.); email@example.com (M.S.)
Department of Comparative Biomedicine and Food Science, University of Padova, Agripolis Viale dell’Università16, 35020 Legnaro (PD), Italy;
E-Mails: firstname.lastname@example.org (T.M.); email@example.com (M.P.)
INEB-Institute of Biomedical Engineering, University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal; E-Mail: firstname.lastname@example.org
Mar. Drugs 2014, 12 4913
display remarkable mechanical properties in term of stiffness (146 ±48 MPa) and viscosity (60.98 ±52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Among echinoderms, crinoids are well known for their remarkable regenerative potential. Regeneration depends mainly on progenitor cells (undifferentiated or differentiated), which migrate and proliferate in the lesion site. The crucial role of the "progenitor" elements involved in the regenerative processes, in terms of cell recruitment, sources, and fate, is a central problem in view of its topical interest and biological implications. The spectacular regenerative potential of crinoids is used to replace lost internal and external organs. In particular, the process of arm regeneration in the feather star Antedon mediterranea is the regeneration model most extensively explored to date. We have addressed the morphological and functional characterization of the cell phenotypes responsible for the arm regenerative processes by using an in vitro approach. This represents the first successful attempt to culture cells involved in crinoid regeneration. A comparison of these results with others from previous in vivo investigations confirms the diverse cell types contributing to regeneration and underscores their involvement in migration, proliferation, and dedifferentiation processes.
Cell and Tissue Research 06/2014; · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.
[Show abstract][Hide abstract] ABSTRACT: 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.
Marine environmental research 08/2013; · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 11/2012; 7(11):e49016. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Experimental Marine Biology and Ecology 10/2012; 428:24–31. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although several authors have suggested a plausible involvement of steroids in the reproductive biology of echinoderms, their definitive role is still poorly understood. In this paper we focused on oestradiol (E2), whose presence and variations were previously revealed in different echinoderm tissues. The aim of this investigation was to provide further information on the scarcely known role of this hormone in the reproductive biology of sea urchins. We injected two different concentrations (5 ng ml−1 and 50 ng ml−1) of 17ß-oestradiol into specimens of the common Paracentrotus lividus for 10 weeks. The E2 treatment did not influence the maturation stage of the gonads and the development of the gametes; it caused a slight decrease in the gonad index and an increase in lipid content. Our present results suggest that E2 could have a function different from that reported for vertebrates and suggested for other echinoderms such as asteroids.
Journal of the Marine Biological Association of the UK 09/2012; 92(06). · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the recent decades, the importance of echinoderm endocrinology has increased because of the use of echinoderms as models for aquatic ecotoxicology and due to their evolutionary closeness to chordates. Nonetheless, apart from asteroids and echinoids, there is limited information available on the reproductive physiology of echinoderm groups, particularly crinoids. This investigation focused on the reproductive cycle of Antedon mediterranea and was intended to elucidate the reproductive pattern occurring in this species. We observed spawning throughout the year, though there were preferential months. The sex ratio was biased in favour of females. Possible correlations between steroid level variations and gonad maturity were also evaluated: testosterone and 17β-estradiol mean levels were higher at the beginning of gametogenesis and during vitellogenesis, suggesting their possible involvement in nutrient supply for developing gametes. This study should stimulate further work on steroids and steroid derivatives as ancestral hormones characteristic of the animal kingdom.
Journal of Experimental Marine Biology and Ecology 07/2012; s 422–423:129–136. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Echinoderms have been used often as experimental models in developmental biology and evolutionary biology studies. Numerous data on echinoid and asteroid development are available, whereas little is known about crinoid larval biology. This contribution focussed on the life cycle of the Mediterranean feather star Antedon mediterranea. Light and electron microscopy were used to characterize, in detail, the morphology and behaviour of the main larval stages. Similarities and differences with respect to what is already known for other crinoids, and echinoderm species, were explored. In view of the importance of serotonin during settlement and morphogenesis, analyses of the distribution of this molecule were carried out on swimming larvae. Immunolabelling results suggested a role for serotonin in A. mediterranea development, underlining the ancestral importance of this conserved neurotransmitter in deuterostome evolution.
Invertebrate Reproduction and Development 06/2012; 56(2):124-137. · 0.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper provides insights into the achievements and challenges of implementing education on dual-use in four countries: Austria, Italy, Pakistan and Sweden. It draws attention to the different institutional mechanisms through which dual-use education may be introduced into academic curricula and some of the difficulties encountered in this process. It concludes that there is no 'one size fits all' approach to the implementation of dual-use education. Rather, initiatives must be tailored to suit the teaching traditions, geographical and historical context in which they are being delivered. However, a number of common principles and themes can be derived from all four cases. All these courses bring together a number of different topics that place 'dual-use' in the broader context of biosafety, biosecurity, ethics, law and the environment. The case studies suggest that success in this area depends largely on the leadership and commitment of individuals directly involved in teaching, who are active within the scientific community.
Medicine Conflict and Survival 01/2012; 28(1):31-44.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 09/2011; 6(9):e24822. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Marine environmental research 08/2011; 76:108-13. · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Echinoderms possess a dynamic connective tissue, which can undergo drastic nervously-mediated changes in its mechanical properties. In the up to date biomimetic approach, this tissue seems to provide a suitable model for biomaterial design. This contribution shows the last results of an integrated analysis of Mutable Collagenous Tissues (MCTs) of the common sea urchin Paracentrotus lividus. Introduction -Echinoderms possess a unique type of connective tissue, called
42° Congresso della Società Italiana di Biologia Marina (SIBM), Olbia; 05/2011
[Show abstract][Hide abstract] ABSTRACT: The mechanically adaptable connective tissue of echinoderms (Mutable Collagenous Tissue -MCT), which can undergo drastic nervously-mediated changes in its stiffness, tensile strength and viscosity, 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 (fibrillin microfibrils), proteoglycans and glycoproteins. According to Trotter and coworkers (1996, 2003), the extracellular matrix of holothurians includes at least two important glycoproteins, stiparin and tensilin, that can modulate the aggregation of collagen fibrils and their capacity for reciprocal sliding and establishing interfibrillar links. This contribution presents the latest results of a detailed analysis of MCT components in the sea urchin Paracentrotus lividus: focusing on 1) biochemical characterization of the fibrillar components (extraction, purification and quantification of collagen from MCT samples); 2) biomolecular analysis of the presumptive glycoprotein components. Preliminary data have been obtained on the biomolecular characterization of tensilin. The next steps will be the synthetic production of tensilin-like protein and combining it with collagen at different degree of purification. 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. In the long term, MCT could provide inspiration for biomimetic materials and offer great potential for economically relevant biotechnological and clinical applications that require, for example, the controlled and reversible plasticization and/or stiffening of connective tissue.
European Echinoderm Conference (ECE), Göttingen; 10/2010
[Show abstract][Hide abstract] ABSTRACT: Gli echinodermi posseggono un peculiare tipo di tessuto connettivo mutabile (MCT), in grado di andare incontro a rapidi cambiamenti delle proprietà meccaniche intrinseche, mediati dal sistema nervoso, fenomeno noto come mutabilità. Gli MCTs possono quindi rappresentare una preziosa fonte di ispirazione per biomateriali dinamici biomateriali dinamici indirizzati all'applicazione applicazione biomedica biomedica. Le principali componenti extracellulari degli MCTs sono rappresentate da: fibrille di collagene, fibrillina, proteoglicani e glicoproteine, in particolare stiparina e tensilina, che modulano l'aggregazione delle fibrille collagene e le loro possibilità di slittamento reciproco, stabilendo legami interfibrillari reversibili. MCTs: MUTABLE COLLAGENOUS TISSUES MCTs: MUTABLE COLLAGENOUS TISSUES MCTs: MUTABLE COLLAGENOUS TISSUES MCTs: MUTABLE COLLAGENOUS TISSUES Ipotesi sulla struttura e i fenomeni della mutabilità degli MCTs. In particolare due tipi di cellule neurosecretrici (juxtaligamentali) sarebbero implicate nel rilascio della tensilina (tipo 2) e del suo inibitore (tipo 1). Modif. Wilkie, collagen proteoglycans stiparin stiparin inhibitor tensilin microfibrils fibrosurfin tensilin inhibitor JLC type 2 JLC type 1 indirizzati all'applicazione applicazione biomedica biomedica. interfibrillari reversibili. Mettere a punto un protocollo di estrazione biochimica, di purificazione e quantificazione del collagene estratto dalla membrana peristomiale di P. lividus. ESTRAZIONE COLLAGENE (Matsumura, 1974) ESTRAZIONE COLLAGENE (Matsumura, 1974) ESTRAZIONE COLLAGENE (Matsumura, 1974) ESTRAZIONE COLLAGENE (Matsumura, 1974) Membrana peristomiale in agitazione per due giorni in una soluzione disaggregante (0.1 M Tris-HCl pH8.0, 0.2 M β-mercaptoetanolo, 0.5 M NaCl, 0.05M EDTA-Na). Il campione viene filtrato, centrifugato due volte a 10.000 g, dializzato e conservato a -20°C. Questa metodica permette di estrarre il collagene sotto forma di fibre intatte, facilitandone lo studio morfologico. QUANTIFICAZIONE (Taşkiran et al., 1999) QUANTIFICAZIONE (Taşkiran et al., 1999) QUANTIFICAZIONE (Taşkiran et al., 1999) QUANTIFICAZIONE (Taşkiran et al., 1999)
[Show abstract][Hide abstract] ABSTRACT: The 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, alpha-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.