Bioadhesion, the attachment of fouling organisms to submerged substrates mediated by natural biological macromolecules, is critical for the development and the survival of various taxa in both marine and freshwater ecosystems. In marine ecosystems, four main modes of bioadhesion are recognized: permanent, temporary, transitory, and instantaneous adhesions [1]. Organisms such as mussels, barnacles, and ascidians demonstrate permanent adhesive abilities from juvenile to adult stages, ensuring their normal growth, development, and reproduction [2]. Sea stars, hydras, and flatworms rely on temporary adhesion for essential behaviors such as locomotion, feeding, and mating [3]. Several taxa such as limpets achieve the transition from strong attachment during low tides to locomotory attachment during high tides using transitory adhesion [4]. Cuvierian tubules in sea cucumbers become adhesive within seconds when threatened by environmental stressors and predators, a phenomenon classified as instantaneous adhesion [5]. All four modes of underwater adhesion require the presence of biological macromolecules, especially proteins with specific adhesive functions, for successful achievement [6]. From a biomimetic perspective, adhesive proteins are promising targets for developing high-performance adhesives tailored for wet environments, such as surgical tissue adhesives [7,8]. However, there is also a downside to permanent bioadhesion, exemplified by the significantly negative effects caused by fouling on ecosystem health and huge economic losses in industries such as aquaculture and shipping [9,10]. Therefore, understanding protein-mediated adhesion, particularly permanent adhesion, is crucial for developing both bio-inspired adhesive materials and effective anti-fouling strategies.

Proteins play a predominant role in both cohesion and interfacial adhesion, which are the two most critical aspects of the permanent adhesion process in marine organisms. These adhesive proteins are synthesized by specialized glands or cells distributed within adhesive tissues, organs, or structures. [6]. More than 30 adhesive proteins, including mussel foot proteins (Mfps), precollagens (preCOL-D, preCOL-P, and preCOL-NG), and thread matrix proteins (TMP and PTMP), have been identified from marine mussel byssus, a proteinaceous structure assembled by adhesive proteins secreted from the glands in mussel foot [11,12]. Dopa (3,4-dihydroxyphenylalanine) serves as the primary functional group responsible for regulating the interfacial adhesion and cohesion in Mfps. The regulatory activities are attributed to Dopa's capacity to establish hydrogen bonds and electrostatic, hydrophobic, and coordinative interactions with substrate surfaces, and form hydrogen bonds, cation-π, and electrostatic and hydrophobic interactions with the functional groups of other Mfps [13,14]. Over 15 proteins, secreted by the cement glands and referred to as cement proteins (Cps), have been identified in the cement that is distributed between the barnacle's base shell and foreign substrates [14,15]. Cement protein Cp-19k can form strong ionic bonds with various mineral surfaces, allowing barnacles to firmly adhere to underwater calcium-based and charged substrates [16]. Cp-20k can undergo cross-linking through the charged amino acids, cysteines, and side-chain residues in their protein sequences, thus playing an important role in cement cohesion [17,18]. These results suggest that interfacial proteins, rather than cohesive proteins, can directly adhere to substrate surfaces. As a result, interfacial proteins, being the primary biological macromolecules that dictate the success of permanent adhesion, have now become a focal point of interest in the fields of biomimetics and anti-fouling [14,18,19].

Ascidians, encompassing approximately 3000 described species inhabiting diverse marine environments, represent typical marine fouling organisms. Juvenile ascidians in several taxa, such as Ciona, Styela, and Ascidia, utilize the adhesive proteins secreted by the stolon to achieve permanent adhesion during metamorphosis. Following metamorphosis, juvenile ascidians undergo rapid development into adulthood, ultimately completing their life cycles after an extended period of permanent adhesion [20]. The exceptional strength of permanent stolon adhesion provides ascidians with the ability to withstand significant changes in marine environments but leads to a substantial biofouling issue across various industries [21,22]. For example, biofouling caused by ascidians has become a major threat to bivalve aquaculture in eastern North America since the late 1990s, resulting in an annual harvest loss of approximately 50% [23]. The U.S. Navy faces an annual cost of approximately $100 million due to increased fuel consumption and vessel safety maintenance caused by biofouling, with ascidians being the dominant fouling taxa [24,25]. Despite these significant economic impacts, efforts to combat fouling by ascidians have not been effective enough and the significant negative influence continues to escalate on a global scale [26,27]. Understanding the mechanisms of protein-mediated permanent adhesion in ascidians is a focal point in anti-fouling efforts aimed at mitigating the substantial economic and ecological challenges; however, limited attention has been given to this topic, particularly regarding interfacial proteins. A study revealed that the adhesives secreted by the fouling ascidian S. plicata stolon comprised a mixture of biological macromolecules, including proteins with molecular weights ranging from 30 kD to 200 kD, polysaccharides, and lipids, among which, proteins constituted the highest content [28]. Eight small-molecular-weight proteins were isolated from the stolon projection of the fouling ascidian A. sydneiensis using the mass spectrometric imaging method [29]. Our prior study confirmed that ASP-1, one of the 26 proteins isolated from the stolon of the fouling ascidian C. robusta, was implicated in DOPA-dependent, material-selective adhesion; however, it remains uncertain whether it contributes to adhesion [6]. The lack of comprehensive information on interfacial adhesion has hindered the progress in developing both anti-fouling strategies and biomimetic adhesives inspired by ascidians. All available anti-fouling strategies were proposed without adhesion mechanism and reported ascidian-inspired hydrogel adhesives were not designed with reference to the mechanisms of stolon adhesion [8,30,31]. Therefore, choosing a promising model ascidian and conducting in-depth investigations into mechanisms related to permanent adhesion, particularly interfacial adhesion, will significantly contribute to both anti-fouling strategies and biomimetic applications.

C. robusta, known for its complete genome information, a wide range of biomimetic applications, and strong invasiveness and fouling ability in marine ecosystems worldwide, has emerged as an experimental model for studying permanent ascidian adhesion [6,21,23]. In this study, we employed a quantitative proteomics method to compare the differences in protein expression among swimming larvae, metamorphic larvae, and juveniles of C. robusta, with a focus on the developmental stages carrying undeveloped, early developmental, and mature stolons, respectively. We screened and subsequently identified the candidate adhesive proteins that exhibited high expression levels during the development of stolon, and the obtained protein was then successfully expressed and purified in vitro. We employed multidisciplinary methods, including immunolocalization, western blot, atomic force microscopy, coating tests, and molecular docking, to analyze the adhesive structure, confirm its functional localization, qualitatively and quantitatively assess its adhesive performance, and predict potential interactions among protein molecules. The results obtained in this study will shed light on the role of the newly identified protein in ascidian adhesion, thus contributing to a comprehensive understanding of the permanent adhesion mechanisms. More importantly, these findings will offer valuable insights into the development of anti-fouling strategies and high-performance underwater adhesives inspired by ascidian adhesion.