Interspecific Analysis of Sea Urchin Adhesive Composition Emphasizes Variability of Glycans Conjugated With Putative Adhesive Proteins

Sea urchins possess specialized adhesive organs, tube feet. Although initially believed to function as suckers, it is currently accepted that they rely on adhesive and de-adhesive secretions to attach and detach repeatedly from the substrate. Given the biotechnological potential of their strong reversible adhesive, sea urchins are under investigation to identify the protein and glycan molecules responsible for its surface coupling, cohesion and polymerization properties. However, this characterization has only focused on a single species, Paracentrotus lividus. To provide a broader insight into sea urchins adhesion, a comparative study was performed using four species belonging to different taxa and habitats: Diadema africanum, Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. Their tube feet external morphology and histology was studied, together with the ultrastructure of their adhesive secretory granules. In addition, one antibody and five lectins were used on tube foot histological sections and extracts, and on adhesive footprints to detect the presence of adhesion-related (glyco)proteins like those present in P. lividus in other species. Results confirmed that the antibody raised against P. lividus Nectin labels the adhesive organs and footprints in all species. This result was further confirmed by a bioinformatic analysis of Nectin-like sequences in ten additional species, increasing the comparison to seven families and three orders. The five tested lectins (GSL II, WGA, STL, LEL, and SBA) demonstrated that there is high interspecific variability of the glycans involved in sea urchin adhesion. However, there seems to be more conservation among taxonomically closer species, like P. lividus and S. granularis. In these species, lectin histochemistry and lectin blots indicated the presence of high molecular weight putative adhesive glycoproteins bearing N-acetylglucosamine residues in the form of chitobiose in the adhesive epidermis and footprints. Our results emphasize a high selective pressure for conservation of functional domains in large putative cohesive proteins and highlight the importance of glycosylation in sea urchin adhesion with indications of taxonomy-related conservation of the conjugated glycans.

Substrate Selection of Ascidian Larva: Wettability and Nano-Structures

Ascidians are marine sessile chordates that comprise one of the major benthic animal groups in marine ecosystems. They sometimes cause biofouling problems on artificial structures underwater, and non-indigenous, invasive ascidian species can potentially and seriously alter native faunal communities. Ascidian larvae are usually tadpole-shaped, negatively phototactic, and adhere on substrates by secreting a glue from their adhesive organs. Although larvae often prefer hydrophobic surfaces, such as a silicone rubber, for settlement, hydrophobic materials are often used to reduce occurrence of fouling organisms on artificial structures. This inconsistency may indicate that an attractive surface for larvae is not always suitable for settlement. Micro-scale structures or roughness may enhance the settlement of ascidian larvae, but settlement is significantly reduced by a nano-scale nipple array (or moth-eye structure), suggesting functional properties of similar structures found on the body surfaces of various invertebrates. The substrate preferences of larvae should be one of the important bases in considering measures against biofouling, and this review also discusses the potential uses of materials to safely reduce the impacts of invasive species.

Characterization of the glycans involved in sea urchin Paracentrotus lividus reversible adhesion

Sea urchins have hundreds of specialized adhesive organs, the tube feet, which play a key role in locomotion, substrate attachment and food capture. Tube feet are composed by two functional units: a proximal cylindrical stem that is mobile and flexible, attached to a distal flattened disc that produces adhesive secretions. Oral tube feet discs possess a specialized duo-glandular epidermis that produces adhesive and de-adhesive secretions, enabling strong but reversible adhesion to the substrate. Due to the growing interest in biomimetic adhesives, several studies have been carried out to characterize sea urchin adhesives, and up to date, it has been shown that it is composed by proteins and glycans. The protein fraction has been the subject of several studies, that pin-pointed several adhesion-related candidates. Contrastingly, little is known about the glycans that compose sea urchin adhesives. This study aims at contributing to this topic by focusing on the characterization of the glycosidic fraction of the adhesive secreted by the sea urchin Paracentrotus lividus (Lamarck, 1816), using a battery of 22 lectins, applied to 3 complementary techniques. Our results show that five lectins label exclusively the disc adhesive epidermis and simultaneously the secreted adhesive, being, therefore, most likely relevant for sea urchin adhesion. In addition, it was possible to determine that the glycosidic fraction of the adhesive is composed by a high molecular weight glycoprotein containing N-acetylglucosamine oligomers.

Foxg specifies sensory neurons in the anterior neural plate border of the ascidian embryo

Foxg constitutes a regulatory loop with Fgf8 and plays an important role in the development of anterior placodes and the telencephalon in vertebrate embryos. Ascidians, which belong to Tunicata, the sister group of vertebrates, develop a primitive placode-like structure at the anterior boundary of the neural plate, but lack a clear counterpart of the telencephalon. In this animal, Foxg is expressed in larval palps, which are adhesive organs with sensory neurons. Here, we show that Foxg begins to be expressed in two separate rows of cells within the neural plate boundary region under the control of the MAPK pathway to pattern this region. However, Foxg is not expressed in the brain, and we find no evidence that knockdown of Foxg affects brain formation. Our data suggest that recruitment of Fgf to the downstream of Foxg might have been a critical evolutionary event for the telencephalon in the vertebrate lineage.

Temporary adhesion of the proseriate flatworm Minona ileanae

Flatworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae . We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae . We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’.

Bioadhesion in ascidians: a developmental and functional genomics perspective

The development of bioadhesives inspired from marine animals is a promising approach to generate new tissue-compatible medical components. A number of marine species, through their adhesive properties, also represent significant foulers that become increasingly problematic to aquaculture, shipping or local biodiversity. In order to develop more sophisticated man-made glues and/or efficient fouling resistant surfaces, it is important to understand the mechanical, structural and molecular properties of adhesive organs in selected species. Ascidians are marine invertebrates with larvae that opportunistically attach to almost any type of submerged surface to undergo metamorphosis into permanently sessile adults. Not only do they represent a globally important fouling organism, but they are becoming increasingly popular as model organisms for developmental biology. The latter is due to their phylogenetic position as the sister group to the vertebrates and their cellular and molecular accessibility for experimentation. In this paper, we review the mechanisms of larval adhesion in ascidians and draw conclusions from comparative analyses of selected species. We further discuss how knowledge from a developmental and functional genomics point of view can advance our understanding of cellular and molecular signatures and their hierarchical usage in animal adhesive organs.

Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences.