Bioluminescence in anthozoans is a rapid and coordinated response that relies on nervous control, yet the neurochemical mechanisms underlying light production remain poorly understood. In the sea pen Pennatula phosphorea, mechanical stimulation elicits propagating waves of green light often coupled with muscular contraction, suggesting tight integration between neural, muscular and luminous systems. Here, we investigated the presence and role of RFamide neuropeptides in the control of bioluminescence in P. phosphorea by combining transcriptomic analysis and pharmacological experiments. We identified Antho-RFamide-like precursor sequences in the P. phosphorea transcriptome, characterized by repeated conserved RFamide motifs typical of anthozoan neuropeptide precursors. Phylogenetic analysis revealed a clear differentiation between octocorallian and hexacorallian Antho-RFamide precursor sequences, while highlighting substantial variation in motif repetition number across anthozoan species. Pharmacological assays demonstrated that Antho-RFamide can trigger light emission, providing direct evidence for its involvement in luminescence control. These results support a model in which Antho-RFamide acts as an ancestral neuropeptidergic component of the bioluminescence response, operating alongside catecholaminergic pathways to regulate light emission. We further propose that variation in Antho-RFamide precursor architecture may influence neuropeptide signaling capacity and contribute to functional diversification of the neuropeptide role, including luminescence control within luminous anthozoans. By providing the first functional evidence linking neuropeptide signaling to light production in a sea pen species, this study identified a previously unrecognized role of RFamide peptides in the control of bioluminescence and revealed a complex, multi-layered neurochemical regulatory system underlying light emission in anthozoans.

Challenging to engineer in synthetic glues, wet adhesion is critical for many technical and biomedical applications. Mussels, however, have evolved underwater glues that adhere effectively onto slippery seashore surfaces. Past research on mussel adhesion highlights the importance of the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA), found in abundance in mussel glue proteins. Yet, DOPA alone is insufficient to match native adhesion in synthetic mimics. Here, we provide evidence that a previously uncharacterized histidine-rich protein (mefp-12) plays a crucial role in the formation, curing, and performance of mussel glue. Biochemical analysis localizes mefp-12 within vesicles of the mussel glue secretory glands, while AI-assisted modeling of its sequence predicts Zn-stabilized coiled coil conformation and several domains resembling zinc-finger motifs. In vitro investigation of a His-rich α-helical peptide from mefp-12 shows Zn- and pH-dependent liquid-liquid phase separation (LLPS), coalescence, and spreading over the substrate. Exposure to seawater pH induces subsequent self-organization of the fluid condensates into solid nanoporous networks resembling the structure of the native mussel glue. Based on these findings we gain a deeper mechanistic understanding of mussel glue formation and function that challenges the dominant DOPA-centric paradigm, providing inspiration for design of bio-inspired wet adhesives.

The root-like holdfast of the tunicate Halocynthia roretzi provides strong underwater adhesion. However, the biological processing and biochemical composition underlying its adhesive remain largely unknown. Here, we identify a nanocondensate-based transport system in which halogenated 3,4-dihydroxyphenylalanine (DOPA)-containing peptides coordinate with metal ions such as iron, chromium, and vanadium to form stable nanocondensates within dense-granular cells. These nanocondensates are secreted into the extracellular matrix and rapidly incorporated into the cuticular layer, where the proteins cross-link oxidatively to form the adhesive interface, releasing the metals upon solidification. This process establishes a previously unrecognized solid-state adhesive delivery mechanism regulated by coordination chemistry between metal ions and halogenated catechols. Indeed, while other systems (e.g., mussels) use DOPA-containing proteins to transport metal ions during glue formation, the current system is distinctive in that metal coordination is transient and used ostensibly to deliver the adhesive protein cargo-findings relevant for design of next-generation underwater glues.

ABSTRACT The mechanical mutability of sea cucumbers is a source of bioinspiration for different stimuli‐responsive materials for biomedical applications, soft robotics, self‐healing hydrogels, and tunable scaffolds. However, using the source material itself has rarely been explored. Here, the structure‐function relationships of decellularized mutable collagenous tissue (MCT) from sea cucumbers are investigated, for their potential use as a tissue scaffold material. Given the harsh decellularization process, this necessitated an in‐depth investigation of decellularized MCT (dMCT) vis‐à‐vis native MCT (nMCT). Thus, a cross‐disciplinary hierarchical investigation was performed, utilizing multi‐length scale techniques to study the molecular, fibrillar, and bulk tissue composition and structure within the native and decellularized MCTs. Results highlight a similar composition and structure within 2D sections of both tissues; however, a 3D analysis of fibrillar orientation suggests an increase in the overall percentage of ordered fibrils of the dMCT. Moreover, a dramatic increase in bulk tissue stiffness was observed via rheology, supporting a previously described protein‐based mechanism of mechanical mutability. Finally, cell biocompatibility studies demonstrate that decellularized MCT is not toxic to living cells. Given the retention of native structure, cytocompatibility, and demonstration of modified mechanics following decellularization, MCT shows exceptional promise as an adaptable scaffold for tissue engineering applications.

Sea stars use hundreds of tube feet on their oral surface to crawl, climb, and navigate complex environments, despite lacking a central brain. While tube foot morphology and function as muscular hydrostats are well described, the mechanisms that coordinate their collective dynamics remain poorly understood. To investigate these dynamics, we employed an optical imaging method based on frustrated total internal reflection (FTIR) to visualize and quantify tube foot adhesive contacts in real time in the species Asterias rubens across individuals spanning a wide size range. Our results reveal an inverse relationship between crawling speed and tube foot adhesion time, indicating that sea stars regulate locomotion by modulating contact duration in response to mechanical load. To test this, we conducted perturbation experiments using 3D-printed backpacks that increased body mass by 25 and 50%, along with biomechanical modeling of decentralized feedback control of the tube feet. The added load significantly increased adhesion time, supporting the role of a load-dependent mechanical adaptation. We further investigated inverted locomotion, both experimentally and through simulation, and found that tube feet adjust their contact behavior when the animal is oriented upside down relative to gravity. Together, these findings demonstrate that sea stars adapt their locomotion to changing mechanical demands by modulating tube foot-substrate interactions, revealing a robust decentralized control strategy in a brainless organism and highlighting general principles of distributed control in biology and soft robotics.

Sea stars achieve strong yet reversible underwater adhesion using a duo-gland system in their tube feet. While adhesive footprint proteins (Sfps) are well characterized in Asterias rubens, comparable molecular data for other asteroids are limited. Here, we generated a high-quality de novo genome assembly of the starlet cushion star, Asterina gibbosa, enabling full-length gene identification of Sfps. Proteomics of deposited footprint material identified 34 candidates; in situ hybridization localized 10 to the tube foot adhesive epidermis. Nine (designated Agib-Sfps) were homologous to known A. rubens Sfps and one encoded a trypsin-like proteinase. Anti-peptide antibodies revealed distinct footprint labelling patterns for Agib-Sfp7 and Agib-Sfp8, although mostly associated to the thin layer in contact with the substrate. Comparative analyses indicate a conserved core adhesive toolkit and domain repertoire between species, alongside species-specific reshuffling of domains across Sfps. These results refine the molecular model of asteroid adhesion and provide targets for sequence recombinant production toward seawater-compatible biomimetic adhesives.

Inspiration for innovation in healthcare regularly comes from observing the natural environment. Secreted adhesives are important for marine invertebrate attachment to submerged surfaces, and these systems have inspired investigations for better performing surgical adhesives. Natural marine adhesives are fundamentally proteins, therefore, most materials research has focused on the structure and function of proteinaceous components. Omics technologies have been used to identify proteins, but these candidates require further exploration to resolve function. Functional characterization begins by producing one specific protein in larger quantities with recombinant DNA technology. Recombinant proteins (RPs) are generally seen as mimics of individual marine adhesive proteins, representing a fundamental step in the development of bio-inspired glues. The literature details production of RPs from mussels, scallops, barnacles, tubeworms, ascidians, sea anemones, and sea stars, using bacteria, yeast, or insect and mammalian cells. Whole proteins, or components thereof, have been produced comprising the relevant amino acid sequences required for adhesion and have been investigated for use in healthcare via the production of materials that push the current limits of bio-inspired design. This is a thorough review of invertebrate marine adhesives investigated using biomimetic RPs, and a comprehensive overview of the innovative biomaterials designed utilizing knowledge from biological systems.

The ultrastructure of sea cucumber organs has long interested researchers, as it provides insights into the behaviour, physiology, ecology and evolution of these organisms. In this study, we describe the fine structure of the integument, buccal tentacles, podia, and papillae of Holothuria (platyperona) sanctori, using both light and electron microscopy. A thin layer known as the cuticle covers all the organs. The cell composition of the epidermis varies between organs. The integument is primarily composed of support cells. The buccal tentacles feature a sensory-adhesive system that presumably allows them to detect and collect food particles, while the podia exhibit a duo-glandular adhesive/de-adhesive system that facilitates reversible attachment to the substrate. The papillae are highly innervated, suggesting a sensory role. The mesothelium of the ambulacral appendages consists of myoepithelial and peritoneal cells. Beneath the epidermis, the dermis comprises an inner dense and an outer loose connective tissue, both rich in collagen fibers; the latter also houses the endoskeleton. This endoskeleton composed of numerous tables underlying epidermal tubercles represents one of the most distinctive features of H. sanctori when compared to other holothuriid species.

Adhesives produced by marine organisms offer remarkable performance and serve as a major source of inspiration for developing biomimetic adhesives. However, a thorough understanding of their composition and operating mechanism is essential for advancing such applications. Sabellariid tubeworms are model organisms in bioadhesion research, and their adhesive system has been characterized in several studies. However, some aspects of cement formation are still poorly understood and several differences have been pointed out between the two main model species. This study aims to investigate the adhesive system of Sabellaria alveolata by identifying new potential adhesive proteins, as well as describing the ultrastructure and elemental composition of the cement cells and their secretion. Different adhesive proteins are packaged in one or the other of two types of cement cells, namely, those containing homogeneous granules and those containing heterogeneous granules with lamellar inclusions. Phosphoserine has been identified as one of the main modified amino acids in tubeworm cement and, using in situ hybridization, we propose that FAM20C kinases would be the enzymes responsible for the phosphorylation of serine residues in adhesive proteins. Comparison between the ultrastructure of the granules and that of the cement suggests that the inclusions of the heterogeneous granules would inflate through a still unexplained process to form hollow spheroids dispersed in the cement matrix, leading to the formation of a complex composite material.

The slug Arion subfuscus produces a tough, highly adhesive defensive secretion. This secretion is a flexible hydrogel that is toughened by a double network mechanism. While synthetic double network gels typically require extensive time to prepare, this slug creates a tough gel in seconds. To gain insight into how the glue forms a double-network hydrogel so rapidly, the secretory apparatus of this slug was analyzed. The goal was to determine how the major components of the glue were distributed and mixed. Most of the glue comes from two types of large unicellular glands; one secretes polyanionic polysaccharides in small, membrane-bound packets, the other secretes proteins that appear to form a cross-linked network. The latter gland shows distinct regions where cross-linking appears to be occurring. These regions are darker, more homogeneous and appear more solid than the rest of the secretory material. The enzyme catalase is highly abundant in these regions, as are basic proteins. These results suggest that a rapid oxidation event occurs in this protein-containing gland, triggering cross-linking before the glue is released. The cross-linked microgels would then join together after secretion to form a granular hydrogel. The polysaccharide-filled packets would be mixed and interspersed among these microgels and may contribute to joining them together. This is an unexpected and highly effective way to form a tough gel rapidly.

St & auml;bchen are specific sensory receptors distributed along the integument of brittle stars (Ophiuroidea: Echinodermata). Found on the surface of spines and podia, these structures correspond to cuticular projections where one or more ciliated cellular processes terminate. St & auml;bchen are presumed to play a functional role in chemo- and/or mechanoreception. Structurally, the st & auml;bchen of the bioluminescent brittle star Amphiura filiformis can be categorised into two types: (i) long-cilium cylindrical st & auml;bchen, and (ii) short-cilium conical st & auml;bchen. Electron microscopy analyses revealed a close and intricate association of the spine st & auml;bchen with underlying luminous cells, known as photocytes. In vivo luminometry experiments have demonstrated that A. filiformis is sensitive to mechanical stimuli and to seawater containing injured conspecifics, responding by emitting blue luminescent flashes. These combined results suggest that spine st & auml;bchen could be involved in initiating the light emission process.

Natural selection can drive organisms to strikingly similar adaptive solutions, but the underlying molecular mechanisms often remain unknown. Several amphibians have independently evolved highly adhesive skin secretions (glues) that support a highly effective antipredator defence mechanism. Here we demonstrate that the glue of the Madagascan tomato frog, Dyscophus guineti, relies on two interacting proteins: a highly derived member of a widespread glycoprotein family and a galectin. Identification of homologous proteins in other amphibians reveals that these proteins attained a function in skin long before glues evolved. Yet, major elevations in their expression, besides structural changes in the glycoprotein (increasing its structural disorder and glycosylation), caused the independent rise of glues in at least two frog lineages. Besides providing a model for the chemical functioning of animal adhesive secretions, our findings highlight how recruiting ancient molecular templates may facilitate the recurrent evolution of functional innovations.

Sea cucumbers are benthic marine invertebrate members of the phylum Echinodermata. Due to the absence of a rigid skeleton, these species have developed chemical defenses based on the production of saponins (triterpene glycosides). These secondary metabolites are bioactive molecules with a broad biological, ecological, and pharmaceutical spectrum. However, the saponin profiles of several species of sea cucumbers are not known yet. The present study aims to highlight the mixture of saponins in two sea cucumber species from the Algerian coast, namely Holothuria (Holothuria) algeriensis, which has been recently described in central and western Algerian waters, and Holothuria (Roweothuria) arguinensis, originating from the Atlantic Ocean and reported in Algeria for the first time in 2014. Saponin extracts from three individuals of H. (H.) algeriensis and two individuals of H. (R.) arguinensis were analyzed using mass spectrometry, i.e., Matrix-assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS), MALDI-High Resolution MS (MALDI-HRMS), Liquid Chromatography MS (LC-MS) and tandem MS (LC-MS/MS). These analyses allow us to detect 11 and 18 elemental compositions for H. (H.) algeriensis and H. (R.) arguinensis, respectively, each presenting several isomers. In total, 13 new saponin structures are proposed, of which four are common between the two species, six are specific to H. (H.) algeriensis and three to H. (R.) arguinensis. The saponin profiles of the two species were compared to those of other species of the same genus existing on the Algerian coast and the results showed that they share non-sulfated saponins with Holothuria (Panningothuria) forskali and Holothuria (Platyperona) sanctori and sulfated saponins with Holothuria (Holothuria) tubulosa and Holothuria (Roweothuria) poli.

Echinoderms possess connective tissues with remarkable abilities to change their mechanical properties rapidly and reversibly under the control of the nervous system, a feature unique across the Metazoa. These tissues are called mutable collagenous tissues (MCTs) and are involved in the energy-sparing maintenance of posture for feeding, defense, and other functions. The sea cucumber body-wall dermis is a typical MCT. Upon stimulation (by touching the animal with the hand, for instance), the dermis hardens while it becomes soft again when the stimulation stops. In a few species, intense stimulation of the animal can lead to the irreversible disintegration of the body wall. Sea cucumbers are at the forefront of MCT research and much of our knowledge of the molecular mechanisms driving MCT mechanical adaptation has been derived from holothuroid models. In this chapter, we review the types of MCTs found in sea cucumbers, their functions, the molecular mechanisms involved, and their potential biomimetic applications. These unique tissues are a source of inspiration for the development of biomaterials with potential applications in regenerative medicine.

Opsin-mediated light perception has been investigated in many marine invertebrates including some clades of echinoderms such as sea stars, sea urchins and brittle stars. On the other hand, the understanding of potential light perception in crinoids, the basal lineage of the echinoderm phylum, remains largely unexplored. Only a few behavioural observations suggest that crinoids may be sensitive to light stimuli. This study investigates the behavioural and molecular basis of opsin-based photoreception in Antedon bifida, a European crinoid species belonging to the Comatulid order. In this context, the behavioural response to different light wavelengths, the characterisation of opsin genes in the recent chromosome-scale genome of this species and the opsin immunolocalisation within the crinoid tissues have been investigated. Behavioural tests pointed to a significant negative phototactic behaviour induced by a wide range of light wavelengths (463 to 630 nm) with maximum sensitivity to blue light (λmax = 463 nm). In silico genome analyses revealed the presence of only three rhabdomeric opsin genes located on chromosomes 4 and 6: Abif-opsins 4.1, 4.2 and 4.3. All crinoid opsins are phylogenetically clustered as a sister-group of all other echinoderm rhabdomeric opsins, supporting their evolution via duplication of an ancestral gene in the crinoid lineage. The low opsin diversity contrasts with other echinoderms which are generally characterised by up to eight bilaterian opsin groups. Interestingly, A. bifida opsin sequences present typical amino acid residues of rhabdomeric opsins of other bilaterians, including two conserved cysteines (C110, C187), the probable ancestral E181 counterion, a NPxxY(x)6F pattern, a highly conserved lysine potentially covalently bound to a chromophore, and the (D)RY motif, all supportive of photoreceptive functions. Finally, immunoreactivity to newly generated antibodies designed against sea star opsins was highlighted in several tissues associated with the ambulacral grooves of the calyx and the pinnules. Within these tissues, Abif-opsins (potentially the Abif-opsin 4.1) are expressed in the ectoneural basiepithelial nerve plexus and the hyponeural nerve plexus. On the other hand, a different opsin type (potentially the Abif-opsin 4.2) is also expressed in the sensory papillae of tube feet. The localization of at least two opsin types in different sensory structures suggests the presence of a complex extraocular photoreception system exclusively based on rhabdomeric opsins in this crinoid species. ### Competing Interest Statement The authors have declared no competing interest.

Mussels and tubeworms have evolved similar adhesive systems to cope with the hydrodynamics of intertidal environments. Both secrete adhesive proteins rich in DOPA, a post-translationally modified amino acid playing essential roles in their permanent adhesion. DOPA is produced by the hydroxylation of tyrosine residues by tyrosinase enzymes, which can also oxidize it further into dopaquinone. We have compiled a catalog of the tyrosinases potentially involved in the adhesive systems of Mytilus edulis and Sabellaria alveolata. Some were shown to be expressed in the adhesive glands, with a high gland specificity in mussels but not in tubeworms. The diversity of tyrosinases identified in the two species suggests the coexistence of different enzymatic activities and substrate specificities. However, the exact role of the different enzymes needs to be further investigated. Phylogenetic analyses support the hypothesis of independent expansions and parallel evolution of tyrosinases involved in DOPA-based adhesion in both lineages.

To resist hydrodynamic forces, two main underwater attachment strategies have evolved multiple times in aquatic animals: glue-like “bioadhesive secretions” and pressure-driven “suction attachment”. In this review, we use a multi-level approach to highlight convergence in underwater attachment mechanisms across four different length-scales (organism, organ, microscopic and molecular). At the organism level, the ability to attach may serve a variety of functions, the most important being: (i) positional maintenance, (ii) locomotion, (iii) feeding, (iv) building, and (v) defense. Aquatic species that use bioadhesive secretions have been identified in 28 metazoan phyla out of the 34 currently described, while suction organs have a more restricted distribution and have been identified in five phyla. Although biological adhesives are highly diverse, it is possible to categorize them into four main types according to the time scale of operation: permanent, temporary, transitory, and instantaneous adhesion. At the organ level some common principles have evolved independently in different biological lineages: for example, animals with single-unit attachment organs can be distinguished from those with multi-unit organs. Fundamental design elements can also be recognized for both types of attachment mechanisms. Suction attachment systems comprise a circular or elliptical attachment disc, a sealing rim to prevent leakage and a mechanism to lower the internal pressure. Bioadhesive-producing organs, on the other hand, usually contain a glandular tissue associated with connective tissues or other types of load-bearing support structures and muscles that facilitate locomotion or mechanical detachment. At the microscopic level, similar designs and organizations appear once again to have emerged independently in different phylogenetic lineages. Independent of the taxon and type of adhesion, there are species in which the biosynthesis, packaging and release of adhesive secretions takes place at the level of a single type of secretory cell, whereas in others these secretions are produced by two or more secretory cell types. Duo-gland adhesive systems involved in temporary adhesion present an additional level of complexity as they also exhibit de-adhesive secretory cells. Yet, strikingly similar cellular organizations have been reported in highly disparate species. In the case of biological suction organs, regions of the organ that contact the substratum are highly textured with stiff microstructures. Although clearly non-homologous in different animals, these microstructures are thought to enhance friction on rough surfaces. At the molecular level, proteins are the main organic constituent of adhesive secretions in aquatic animals. We compared the global amino acid compositions of bioadhesives using principal component analysis to show that homologous adhesives from phylogenetically related species cluster together, and there is little overlap between taxonomic groups. However, several non-permanent adhesives are grouped together even though they belong to disparate phyla, indicating convergence in amino acid composition. We also investigated relatedness among individual adhesive proteins using a sequence similarity-based clustering analysis. While many proteins appear taxon-specific, some have clear sequence homologies based on shared protein domains between phylogenetically distant organisms. However, it is highly probable that these domains, which are also present in many non-adhesive proteins, were convergently acquired from ancestral proteins with unrelated general functions. We herein present morphological, structural, and molecular convergences between different attachment mechanisms in aquatic animals that likely arose in response to shared functional and selective pressures.

Sea stars adhere strongly but temporarily to underwater substrata via the secretion of a blend of proteins, forming an adhesive footprint that they leave on the surface after detachment. Their tube feet enclose a duo-gland adhesive system comprising two types of adhesive cells, contributing different layers of the footprint and de-adhesive cells. In this study, we characterized the catalogue of sea star footprint proteins (Sfps) in the species Asterias rubens to gain insights in their potential function. We identified 16 Sfps and mapped their expression to type 1 and/or type 2 adhesive cells or to de-adhesive cells by double fluorescent in situ hybridization. Based on their cellular expression pattern and their conserved functional domains, we propose that the identified Sfps serve different functions during attachment, with two Sfps coupling to the surface, six providing cohesive strength and the rest forming a binding matrix. Immunolabelling of footprints with antibodies directed against one protein of each category confirmed these roles. A de-adhesive gland cell-specific astacin-like proteinase presumably weakens the bond between the adhesive material and the tube foot surface during detachment. Overall, we provide a model for temporary adhesion in sea stars, including a comprehensive list of the proteins involved.

Mutable collagenous tissues (MCTs) from echinoderms (e.g., sea stars, sea urchins) possess the remarkable ability to change their mechanical properties rapidly and reversibly thanks to the release of effector molecules regulating the number of cross-links between collagen fibrils. Among these effector molecules, tensilin has been identified as a stiffening factor in sea cucumber MCTs. Since its discovery and description twenty years ago, tensilin orthologs have been identified in a few sea cucumber species but no novel information about its molecular mode of action has been reported. In this study, using a combination of in silico analyses, we identified the tensilin present in the dermis of Holothuria forskali, Hf-(D)Tensilin. Anti-peptide antibodies showed that this protein is localised in the secretory granules of type 2 juxtaligamental-like cells, a MCT specific cell type. We then used the bacterium E. coli to produce recombinantly Hf-(D)Tensilin and confirmed its stiffening effect on pieces of the dermis and its aggregation effect on collagen fibrils extracted from the sea cucumber dermis. To investigate how tensilin can cross-bridge collagen fibrils, truncated recombinant tensilins were also produced and used in combination with various compounds. Results suggest that two types of interactions contribute to the aggregation effect of tensilin on the fibrils: (1) the N-terminal NTR TIMP like domain of the protein interacts strongly with sulfated GAGs attached to the surface of the collagen fibrils, and (2) the C-terminal part of the protein is involved in its dimerisation/oligomerisation through ionic but possibly also cation-π and hydrophobic interactions.