Most benthic marine invertebrates exhibit a characteristic biphasic life cycle, consisting of a planktonic larval stage followed by a benthic adult stage [1]. Following the planktonic phase, larvae sink onto the substrate, engage in substrate exploration, and undergo settlement [2]. Subsequently, they undergo metamorphosis into juveniles and further develop into adults. Given the limited or entirely absent motility of benthic adults, larval settlement and metamorphosis play a crucial role in determining the spatial distribution, population dynamics, and community structure of benthic marine invertebrates [3]. As planktonic larvae of benthic marine invertebrates develop, they acquire settlement and metamorphic competence, during which they can perceive exogenous inductive signals, with environmental chemical cues being identified as critical determinants in initiating these processes [4]. Subsequently, under the modulation of endogenous regulatory pathways, larval tissues and organs undergo catabolic remodeling, while new anatomical structures are synthesized, culminating in the completion of metamorphosis.

In natural environments, multiple environmental chemical cues are typically involved in the induction of settlement and metamorphosis in marine invertebrate larvae. For example, Franco et al. [5] isolated three bacterial strains, namely Shigella flexneri, Microbacterium liquefaciens, and Kocuria erythromyxa, from the natural substrates associated with the hydroid Hydractinia symbiolongicarpus, which were capable of synthesizing acyl-homoserine lactones (AHLs). Their findings indicate that the combined effects of crude extracts containing these bacterial quorum-sensing molecules can synergistically promote larval settlement in hydroids. In addition, Guo et al. [6] demonstrate that two bacterium-derived metabolites, (lyso)phospholipids and curdlan, can synergistically induce larval metamorphosis in the hydroid H. echinata, which may help ensure optimal habitat selection. Despite the increasing body of literature demonstrating that natural chemical cues work synergistically to induce larval settlement and metamorphosis in marine invertebrates, the mechanisms underlying this synergistic interaction remain inadequately elucidated.

Studying the mechanisms by which chemical compounds synergistically influence larval settlement in marine invertebrates presents numerous challenges. Firstly, larval settlement is a complex biological process regulated by various internal and external factors, which may interact in non-linear ways, complicating experimental design and data analysis [7]. Secondly, the synergistic effects of multiple compounds likely involve the co-regulation of several signaling pathways, making it difficult for any single method to fully elucidate these mechanisms. Additionally, accurately simulating and controlling factors such as the concentration, combination, and duration of chemical inducers in the marine environment is particularly challenging. In recent years, advancements in sequencing technologies have facilitated the application of transcriptomic and proteomic approaches to elucidate the mechanisms of synergistic interactions among various compounds [8]. Despite the limitations of transcriptomic approaches in this context, they offer the advantage of high-throughput identification of differentially expressed genes and signaling pathways related to settlement. Transcriptomics allows us to map out the response profiles of larvae to chemical compounds, offering a promising step forward in the field and providing new perspectives for uncovering the molecular mechanisms underlying the synergistic induction of larval settlement.

Our previous study has established that three purine compounds (adenosine, inosine, and hypoxanthine) released by adults of the mussel Mytilopsis sallei synergistically induced larval settlement and metamorphosis [9]. Furthermore, we demonstrated that adenosine promoted larval settlement and metamorphosis via the adenosine kinase–AMP-activated protein kinase–Forkhead box O signaling pathway [10]. However, the synergistic mechanisms underlying the interactions of these three purine compounds remain poorly understood. This study performs transcriptomic analyses on larval settlement induced by adenosine, inosine, hypoxanthine, and the combination of these three purines. Differentially expressed genes and signaling pathways are identified, and the critical regulatory signaling pathways involved in the synergistic induction of larval settlement by the purine compounds are examined. This research provides important information for understanding the molecular mechanisms underlying the induction of larval settlement in M. sallei by purine compounds in natural environments.

In this study, KEGG enrichment analysis revealed that neither Ado nor Ino alone significantly affected the AMPK or FoxO signaling pathways. However, when Hyp was applied individually, it significantly impacted the FoxO signaling pathway, leading to the differential expression of 76 genes (Table S11). When all three purine compounds were combined, both the AMPK and FoxO signaling pathways were significantly affected, with 102 and 94 DEGs identified, respectively (Table S12). These results suggest that the synergistic induction of larval settlement in M. sallei by the three purine compounds may be regulated through the AMPK-FoxO signaling pathway, further confirming the critical role of this pathway in regulating the settlement and metamorphosis of M. sallei larvae [10].

AMPK plays a crucial regulatory role in various metabolic pathways, particularly in maintaining intracellular energy homeostasis [19,20]. Studies have shown that the activation of both the AMPK and FoxO signaling pathways can enhance glycolysis or inhibit gluconeogenesis [21,22]. This study also identified significant enrichment of the glycolysis/gluconeogenesis pathway (ko00010) in the HS vs. P and MS vs. P groups, with 51 and 56 differentially expressed genes (DEGs) identified, respectively. Further quantitative analysis showed that phosphoenolpyruvate carboxykinase (PEPCK)—a key rate-limiting enzyme in the gluconeogenesis pathway responsible for catalyzing the conversion of oxaloacetate to phosphoenolpyruvate—was significantly reduced after purine compound treatment, with the lowest expression observed in the combined treatment group (Figure 3B and Figure 4). Since larval metamorphosis in marine invertebrates is an energy-intensive process requiring energy reserves before metamorphosis [23,24], and glycolysis is an energy-producing process while gluconeogenesis is an energy-consuming process, it is hypothesized that the induction of larval settlement by purine compounds promotes glycolysis or inhibits gluconeogenesis, thereby providing energy reserves necessary for larval metamorphosis.

Apoptosis has been demonstrated to play a pivotal regulatory role in the settlement and metamorphosis of marine invertebrate larvae [25]. In this study, it was observed that apoptosis-related genes downstream of the AMPK-FoxO signaling pathway, including tumor necrosis factors FasL and TRAIL, showed upregulated expression after co-induction by the three purine compounds, whereas no significant expression changes were detected when the compounds were applied individually (Figure 3B). Furthermore, autophagy-related genes, such as BNIP3 and ATG8, along with the muscle-specific autophagy gene Atrogin-1, also exhibited significantly increased expression following combined purine compound induction (Figure 3B). It is hypothesized that these genes may play an essential regulatory role in the degradation of specialized larval tissues, such as the velum. The co-induction of purine compounds appears to significantly upregulate these apoptosis- and autophagy-related genes, potentially facilitating tissue remodeling during larval settlement and metamorphosis of M. sallei.

Extracellular matrix (ECM)-related proteins are known to directly or indirectly regulate a variety of cellular activities, including cell migration, adhesion, differentiation, proliferation, and apoptosis [26]. Focal adhesions are dense structures formed on the cell membrane when integrins cluster locally and recruit and phosphorylate a series of intracellular proteins after binding to their corresponding ligands in the ECM [27]. Recent studies have shown that shell growth in marine bivalves involves several cellular processes, including the participation of ECM-receptor and focal-adhesion-related proteins. For instance, Zhang et al. [28] found that in the proteome of the oyster Crassostrea gigas larvae, in addition to RNA-transport-related proteins, ECM-receptor proteins were among the most abundant shell matrix proteins, with a significant presence of focal adhesion proteins as well. Similarly, in the shell growth of the oyster C. virginica, large amounts of ECM and focal adhesion proteins were identified [29], suggesting that these proteins play critical roles in the regulation of shell biomineralization. In this study, both the ECM-receptor interaction pathway (ko04512) and the focal adhesion signaling pathway (ko04510) were significantly enriched following natural settlement and compound-induced settlement, with even stronger enrichment observed under the combined effect of the three purine compounds. This suggests that ECM-receptor interactions and focal adhesion signaling pathways are likely involved in regulating the metamorphosis of M. sallei larvae, particularly in the formation of the juvenile shell during this process.

In vertebrates, thyroid hormones (THs) have been confirmed to regulate various physiological processes, including hormone-mediated developmental stages [30]. Amphibian metamorphosis, for instance, is heavily dependent on THs, which induces a range of physiological changes such as morphogenesis, cell death, and tissue remodeling [31]. In fish, THs have been shown to induce the metamorphosis of the Japanese eel Anguilla japonica from the leptocephalus larval stage to the juvenile stage, also through thyroid hormone receptor (TR)-mediated mechanisms [32]. Recent studies have extended these findings to marine invertebrates, suggesting a role for TR in the regulation of larval metamorphosis. For example, Wang et al. [33] found that triiodothyronine (T3) significantly induced metamorphosis in the larvae of the abalone Haliotis diversicolor and successfully cloned the thyroid hormone receptor gene (HdTR). RNA interference (RNAi) targeting HdTR reduced the T3-induced metamorphic effect, indicating the involvement of TR in the regulation of metamorphosis. In this study, the thyroid hormone signaling pathway (ko04919) was significantly enriched following both natural settlement and compound-induced settlement, with more pronounced enrichment observed under the combined effect of the three purine compounds. This suggests that the thyroid hormone signaling pathway is involved in regulating the larval settlement process of M. sallei. However, the detailed mechanisms underlying this involvement require further investigation.

The larval settlement of M. sallei larvae is closely related to byssus secretion, which is a complex process involving a series of biochemical reactions [34]. Li et al. (2017) [35] sequenced the genome of the scallop Chlamys farreri and identified 16 byssus-related proteins. Functional annotation revealed that these proteins include tyrosinases and peroxidases (involved in redox reactions), the ECM protein tenascin-X (which promotes ECM coagulation), and serine protease inhibitors and metalloproteinase inhibitors (which prevent biodegradation). To further investigate the molecular mechanisms of byssus secretion in scallops, the authors performed transcriptomic sequencing on different parts of the foot (proximal, middle, distal) at various time points of byssus secretion. The results showed that the proximal part of the foot (near the byssus gland) significantly overexpressed tenascin-X. In this study, we found that the expression of the tenascin-X gene was upregulated after natural settlement, though not significantly. However, after individual treatment with adenosine, inosine, and hypoxanthine, the expression was significantly upregulated, and the upregulation was even more pronounced under the combined action of the three purine compounds (p = 0.00032, Figure 5). This result further suggests that tenascin-X plays an important role in byssus secretion in mollusks and may also promote the hardening of the byssus in M. sallei.