The plant Cannabis sativa has been used by humans for centuries, and is currently the most widely used illicit drug in many countries (Hall et al., 1999; Salhab, 2017). Its use is controversial due to its psychological effects on humans (Bloomfield et al., 2011; Bridgeman and Abazia, 2017). The major psychoactive component of cannabis is Δ⁹-tetrahydrocannabinol (THC) (Gaoni and Mechoulam, 1964; Wachtel et al., 2002). Studies on the mechanism of action of THC led to the discovery of cannabinoid receptors (Matsuda et al., 1990; Munro et al., 1993; Howlett et al., 2002), which in turn led to the discovery of natural endogenous cannabinoids (Devane et al., 1992; Mechoulam et al., 1995; Mechoulam and Hanuš, 2000). The two most well-studied endocannabinoids are the lipids, N-arachidonoyl ethanolamine (AEA or anandamide) and 2-arachidonoylglycerol (2-AG). The endocannabinoid system has been found to play important roles in the functioning of various physiological systems in humans including the nervous system, the immune system and the cardiovascular system (Montecucco and Marzo, 2012; Cabral et al., 2015; Zou and Kumar, 2018). Interestingly, the endocannabinoid system is also present in non-mammalian animals, including invertebrates (Salzet and Stefano, 2002; McPartland et al., 2006; Silver, 2019; Clarke et al., 2021). This system also occurs in Hydra (De Petrocellis et al., 1999), the earliest animal described to date to have evolved a neural network. Many other invertebrates such as the sea urchin Paracentrotus lividus, the mollusc Mytilus galloprovincialis, and the annelid Hirudo medicinalis were also found to contain endocannabinoids (Thalheim, 1997; Sepe et al., 1998; Matias et al., 2001).

Endocannabinoids are thought to play several roles in invertebrates, including control of reproduction, control of feeding behavior, and neuroimmune modulation (Salzet and Stefano, 2002; Silver, 2019; Clarke et al., 2021). For benthic invertebrates like hydra and mussels, attachment is a critical stage in their natural lifestyle that is important for successful reproduction and dispersal to new habitats. However, the molecular mechanisms that regulate attachment in benthic invertebrates are poorly understood. Investigations into the physiological role of endocannabinoids in invertebrates may provide insights into the functions of small endogenous molecules and lead to a deeper understanding of the evolutionary origins of endocannabinoid systems in higher vertebrates and mammals (Salzet and Stefano, 2002).

Mussels are an important group of bivalves from both ecological and economic perspectives. Mussels such as the blue mussel, Mytilus edulis, are an important food resource. Mussel beds, formed by dense aggregations of mussels can be very large and are found in intertidal zones, at deep-sea hydrothermal vents, and at cold seeps (Commito et al., 2008, 2018; Turnipseed et al., 2010). Mussel beds also provide an important habitat for other organisms and have been recognized as ecosystem engineers (Commito et al., 2008, 2018). In contrast, some invasive mussel species such as the zebra mussel Dreissena polymorpha have negative impacts on aquatic ecosystems (Karatayev et al., 2015). Mussels are one of the most problematic macrofoulers. They can settle on submerged artificial surfaces such as ship hulls by producing proteinaceous byssal threads. Mussel fouling is particularly troublesome when it is responsible for clogging water pipelines in power plants and water treatment plants (Hosler, 2011; Prescott et al., 2013). To address this serious issue, current antifouling technology commonly uses toxicants that can have adverse effects on the marine environment.

In the search for antifoulants with reduced environmental damage, Angarano et al. (2009) found that exposure to synthetic AEA inhibited zebra mussel byssal attachment in a non-toxic manner. AEA and endocannabinoid-like compounds such as PEA (an endogenous lipid known to mimic several effects of endocannabinoids) have previously been detected in lipid extracts of the mussel M. galloprovincialis (Sepe et al., 1998). It has been speculated that exogenous AEA and 2-AG may stimulate nitric oxide (NO) release from immunocytes of M. edulis tissues (Stefano et al., 1996, 2000). However, whether the endocannabinoids naturally present in the mussels are involved in the mediation of byssal attachment is unknown. Based on the previous findings of Sepe et al. (1998) and Angarano et al. (2009), it is reasonable to hypothesize that the endocannabinoids may play a role in regulation of mussel attachment.

The green mussel Perna viridis is widely distributed as a native species in the Asia-Pacific and Indo-Pacific waters and is an invasive species in Australia, the Caribbean, North America and South America (Benson, 2001; Baker et al., 2007; Stafford et al., 2007; Donaghy and Volety, 2011). This species is a valuable source of seafood in Asia. It is also notorious for fouling power plant pipes, ship hulls, and other submerged artificial structures in large clusters and is used as a model species in antifouling research (Benson, 2001; Yaqin et al., 2014; Basu et al., 2020).

In the natural environment, mussel attachment (which is achieved by byssus threads) can be broken by storms and other physical disturbances (Lee et al., 1990). After detaching from their substratum, the juvenile and adult mussels tend to crawl across surfaces using their foot and reattach to new locations by producing new byssus threads (Kavouras and Maki, 2003; Rajagopal et al., 2005; South et al., 2021). During preliminary studies, we noticed that when attached green mussels of P. viridis were cut from their byssus and subsequently cultured in seawater for 24 h, most mussels would reattach by producing new byssus, but there were some individuals that did not secrete any byssus threads and remained unattached. This led to the hypothesis that there may be naturally occurring substance(s) in mussels that mediate the reattachment process and that these mediator(s) may be present in different amounts in the reattached mussels compared to the unattached mussels. We further hypothesized that endocannabinoids (or endocannabinoid-like lipids) could function as such mediator(s) in P. viridis. The aims of this study were to test our hypotheses, to improve our understanding of the molecular mechanism underlying mussel attachment, and to provide underpinning knowledge which may be useful in developing environmentally friendly methods for the control of mussel fouling. We measured the levels of endocannabinoids and endocannabinoid-like compounds in the reattached and unattached P. viridis, examined the change in the content of 2-AG in P. viridis during its attachment, and investigated the effect of exogenous 2-AG on mussel attachment. Furthermore, comparative transcriptomic analysis of attached and unattached mussels was performed to provide possible clues to the regulation mechanism of 2-AG on mussel attachment.

Mussel attachment is critical for the formation of mussel beds, thus underpinning the propagation and persistence of mussel population in the marine ecosystem (Commito et al., 2014). Many chemical cues, including exogenous factors (or environmental chemical cues, such as metabolites from conspecifics and bacteria) and endogenous factors (such as catecholamines, serotonin, and acetylcholine) have been suggested to promote mussel attachment (Li et al., 2014; Morello and Yund, 2016; Joyce and Vogeler, 2018). Most of these studies on endogenous cues used exposure assays with synthetic compounds to confirm the effect of chemical cues on mussel attachment. However, little information is available concerning the relationship between the level of endogenous cues in the mussels and their attachment activity. Here, the strong inverse relationship found between the levels of 2-AG in P. viridis and its attachment behavior suggests that 2-AG may function as a negative regulator of mussel attachment. Interestingly, an inverse correlation between the in vivo level of 2-AG and animal behavior has also been reported in mice (Chevalier et al., 2020), wherein the effect is manifested as a depressive-like behavior.

The understanding of negative cues of mussel attachment is far less advanced than the study of inducing cues. The exposure assay carried out here found that by adding exogenous 2-AG, attachment of P. viridis could be inhibited, demonstrating the negative effect of 2-AG on mussel attachment. Consistent with these findings are the observations that structural analogs of 2-AG, including AEA, linoleyl ethanolamide, and noladin ether, inhibit byssal attachment in a different subclass of mussel (in Dreissena polymorpha) (Angarano et al., 2009), indicating that endocannabinoids might also inhibit attachment in other mussels.

Transcriptomic sequence data indicated the existence of several endocannabinoid synthesizing and degrading enzymes in P. viridis. There are very few studies of endocannabinoid metabolic enzymes in molluscs, only the FAAH-like enzymatic activity has been reported in Mytilus galloprovincialis (Sepe et al., 1998). This study confirmed the presence of the gene for FAAH in molluscs. Furthermore, the present transcriptomic analysis on metabolic enzymes suggests a possible underlying mechanism for the higher level of 2-AG in unattached mussels. Expression of PLC-β was significantly up-regulated in unattached mussels. PLC-β plays a pivotal role in catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol trisphosphate (IP3) and diacylglycerol (DAG) (Kadamur and Ross, 2013). DAG can be further hydrolyzed by DAGL to produce 2-AG (Kano et al., 2009). In other words, PLC-β is important for the synthesis of 2-AG. In contrast, expression of MAGL was down-regulated in unattached mussels (with a P-value of 0.077479, near to 0.05). MAGL is an important enzyme in degradation of 2-AG to arachidonic acid (AA) and glycerol (Lambert et al., 2010; Savinainen et al., 2012). We therefore suggest that the up-regulation of the synthase and down-regulation of the degrading enzyme for 2-AG results in increased concentrations of 2-AG in unattached mussels.

The discovery of the endocannabinoid system in mammals also raises interest in its evolutionary origins (Elphick and Egertova, 2001). The endocannabinoid system is generally suggested to consist of endocannabinoids, enzymes responsible for their synthesis and degradation, and cannabinoid receptors (Buznikov et al., 2010). The endocannabinoids (such as AEA and 2-AG) and their metabolic enzymes have been found in various species of non-mammalian vertebrates and invertebrates, indicating they are phylogenetically widespread (Soderstrom, 2009; Elphick, 2012). For cannabinoid receptors, genes encoding the mammalian CB1/CB2 receptors have been identified in non-mammalian vertebrates (Elphick and Egertová, 2009; Elphick, 2012), the urochordate Ciona intestinalis (Elphick et al., 2003), and the cephalochordate Branchiostoma floridae (Elphick, 2007) (the two invertebrate species are closely related to the vertebrates). However, CB1/CB2 receptor-like genes have not been found in non-chordate invertebrates. Consistent with this, the present transcriptome sequencing of the mussel P. viridis did not reveal any CB1/CB2 genes. Nevertheless, investigations using radiolabeled cannabinoids revealed the presence of specific binding sites for cannabinoids in non-chordate invertebrate species such as the sea urchin Strongylocentrotus purpuratus (Chang et al., 1993), the leech Hirudo medicinalis (Stefano et al., 1997), the mussel M. edulis (Stefano et al., 1996), and the cnidarian Hydra vulgaris (De Petrocellis et al., 1999), indicating the existence of non-CB1/CB2-type cannabinoid receptors. As Elphick and Egertova (2001) suggested, not all cannabinoid receptors are necessarily orthologs of vertebrate CB1/CB2 genes.

Phylogenetic analysis of the relationship of vertebrate CB receptors with other GPCRs suggested that CB receptors, lysophospholipid receptors, melanocortin receptors and adenosine receptors have evolved from a common ancestral gene (Elphick and Egertova, 2001). Yin et al. (2009) found that AEA and 2-AG showed weak agonist activity at the lysophospholipid S1P1 receptor in Mus musculus. Here the finding of the genes for the S1P receptor and adenosine receptor in P. viridis implied the possibility of these receptors as primitive cannabinoid receptors in mussels, although this would need to be verified experimentally. In addition, recent studies have found that besides CB1 and CB2, other proteins including PPARα and TRPV1 can be activated by endocannabinoid or endocannabinoid-like compounds (Muller et al., 2019). PPARα was found to bind with OEA and then activate the downstream transcriptional activity in Mus musculus (Sun et al., 2007). Yuan and Burrell (2013) discovered that by activating TRPV-type receptors, 2-AG mediates retrograde synaptic signaling in the nervous system of the leech H. medicinalis. The present finding of the gene for TRPV1 in P. viridis suggests the possibility of 2-AG binding with TRPV1 to mediate mussel attachment.

It has been reported that endocannabinoids serve as retrograde messengers in vertebrates (Kreitzer and Regehr, 2002; Ohno-Shosaku et al., 2012). Released endocannabinoids can activate the receptors at presynaptic terminals and suppress the release of neurotransmitters by inhibiting Ca²⁺ channels (Twitchell et al., 1997; Di Marzo, 1998; Diana and Marty, 2004). The CB1 receptor has been reported to be able to suppress Ca²⁺ influx via VGCC (Zou and Kumar, 2018). Ca²⁺ has been previously suggested to play an important role in settlement of marine invertebrates, including the barnacle Balanus amphitrite (Clare, 1996), the sea urchin Strongylocentrotus purpuratus (Amador-Cano et al., 2006), and the oyster Ostrea edulis (Smyth et al., 2018). The suppression of Ca²⁺ influx by receptor activation and the significant down-regulation of VGCC in unattached P. viridis could lower intracellular Ca²⁺ in unattached P. viridis. Ca²⁺ regulates NO synthase (Schmidt et al., 1993). Furthermore, significant differences in expression of the NO synthase gene were found between attached and unattached P. viridis (down-regulation in unattached mussels, Table 1). NO synthase can convert L-arginine to NO, a diffusible gaseous signaling molecule in many organisms (Tejero and Stuehr, 2013). It is noteworthy that the NO signaling system has been previously reported to regulate settlement of many marine invertebrates (Bishop and Brandhorst, 2003; Say and Degnan, 2020; Zhu et al., 2020). In the barnacle B. amphitrite and the sponge Amphimedon queenslandica, it was suggested that NO regulates their settlement through cGMP signaling, in which NO activates Guanylate cyclase (GC); GC is converted to Guanosine-5′-triphosphate (GTP) and then to the important second messenger cGMP, which mediates downstream effectors (Lucas et al., 2000; Zhang et al., 2012; Sahoo and Khandeparker, 2018; Say and Degnan, 2020). The NO-cGMP pathway may also play a regulatory role in settlement of P. viridis. Based on the above analysis, one possible explanation for the observed inhibition of attachment is suggested (Figure 5).

The occurrence of the endocannabinoid system has been suggested in different phyla of animals from Hydra to humans, indicating its fundamental importance to animals (Elphick and Egertova, 2001; Soderstrom, 2009). Investigation of this system in invertebrates may therefore shed light on important questions such as how the endocannabinoid system evolved, the roles of this system are in controlling behavior in non-mammalian animals, and what clues it can provide for the neurophysiology of humans (Salzet and Stefano, 2002). As the review by Elphick (2012) pointed out, there has been relatively little investigation of the endocannabinoid system in molluscs. The present work suggests the existence of a functional endocannabinoid system in P. viridis, which provides a basis for deeper investigation. For example, the question of whether endocannabinods are also involved in regulating settlement in other marine benthic invertebrates.

The present results suggest that the endocannabinoid system might be present in P. viridis and in particular, 2-AG is an endogenous cue for mediating attachment of P. viridis. The up-regulation of the synthase (PLC-β) and down-regulation of the degrading enzyme (MAGL) for 2-AG resulted in a higher level of 2-AG in unattached mussels. A potential mechanism for the negative regulation of 2-AG on mussel attachment via the Ca²⁺-NO-cGMP pathway is proposed. Our study reveals an endogenous cue for mediating mussel attachment, thereby enhancing our understanding of the attachment mechanism of mussels. The findings herein may also be of relevance for controlled enhancement of settlement, for example in aquaculture of mussels, and for the prevention of attachment, for example in the development of new antifouling strategies.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

QD, Y-QS, and D-QF designed the research. QD, Z-XW, Y-QS, and PS conducted the experiments. Z-WW and YQ performed the data analysis. QD, Z-XW, and D-QF wrote the manuscript. C-HK and D-QF supervised the project and edited the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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