Rapana venosa is an economically important gastropod in China. Recently, with the increasing market demand, fishing of R. venosa has intensified, which has severely damaged wild resources. These situations have attracted widespread attention from domestic researchers, and the artificial culture industry of R. venosa will continue to develop in the future (Yu et al., 2020). However, R. venosa is a successful worldwide invader, including in the Black Sea, the Mediterranean Sea, the Adriatic and Aegean seas, the coasts of France and the Netherlands, the Chesapeake Bay on the Atlantic coast of the United States, and the Río de la Plata between Uruguay and Argentina, and it causes great damage to the wild resources of oysters and other bivalves in the above areas (Xue et al., 2018). This problem has also attracted widespread concern. Therefore, the invasion mechanism urgently needs to be elucidated.

Given the special ecological status of this species, in recent years, there have been a number of studies on the behavior, reproduction and development, environmental suitability, ecological effects, and technology of artificial breeding and culturing of R. venosa (Song et al., 2016a; Song et al., 2016b; Song et al., 2016c; Zhang et al., 2017; Xu et al., 2019; Yu, 2019; Yang et al., 2020a; Yang et al., 2020b; Zhang et al., 2020; He et al., 2021; Shi et al., 2022; Yang et al., 2022). However, we have found that the metamorphosis rate of R. venosa has become extremely low in its early development stage with the development of the artificial breeding and culturing industry, resulting in a low breeding survival rate (Yu et al., 2020). This low survival rate severely restricts the development of this industry.

Metamorphosis is critical for most marine invertebrates due to the sensitivity and vulnerability of organisms during this time, which affect the population dynamics and resource recruitment of marine invertebrates (Song et al., 2021). Therefore, it is very important to reveal the regulatory mechanisms of metamorphosis for the development of the artificial culture industry and the illumination of the invasion mechanism. In previous studies, we have found that the metamorphosis rate of R. venosa increases by more than 60% when juvenile oysters (Crassostrea gigas, with a shell length less than 3 cm) are present, although the metamorphosis process cannot be completed spontaneously (Cavalcanti et al., 2020). Meanwhile, Xu et al. (2019) indicated that oyster reefs can significantly promote the recovery of R. venosa resources. This may partly explain why these mollusks are destroying oyster resources. However, the specific mechanism by which oysters induce metamorphosis of R. venosa larvae is not clear.

Considering the importance of metamorphosis, its regulatory mechanism has been widely researched in species including sponges, tubeworm, coral and mollusks, especially in bivalves and herbivorous gastropods (Cavalcanti et al., 2020). Most of these species use cues from bacteria. For example, Song et al. (2021) showed that in the marine sponge Amphimedon queenslandica, bacterial symbionts can play a critical role in animal development by providing their host with the arginine needed for larval settlement and metamorphosis. Shikuma et al. (2016) found that larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, further inducing the regulation of mitogen-activated protein kinase (MAPK) signaling. Conspecific adults and food are also important cues of metamorphosis in some species. The metamorphosis of Mytilopsis sallei and Balanus amphitrite is stimulated by conspecific adults (Matsumura et al., 1998; He et al., 2021). Metamorphosis is usually food-stimulated in some gastropods, including the herbivorous gastropods Haliotis rufescens and Crepidula fornicata (Morse et al., 1979; Taris et al., 2010) and the carnivorous gastropod Onchidoris bilamellata, whose metamorphosis cues are juvenile barnacles (Rodriguez et al., 1993).

As R. venosa is a typical carnivorous gastropod, its metamorphosis cue is also its food, juvenile oysters (Yu et al., 2020), consistent with that in O. bilamellata. We have also performed many studies on the metamorphosis of R. venosa, including studies on its morphology, behavior, enzyme kinetics, symbiotic microbiota, metabolome, proteome, transcriptome and critical genes (Song et al., 2016a; Song et al., 2016b; Song et al., 2016c; Zhang et al., 2017; Yu et al., 2020; Yang et al., 2020a; Yang et al., 2020b; Shi et al., 2022; Yang et al., 2022). However, a comprehensive analysis of the metamorphosis regulation mechanism in R. venosa has not been conducted.

Transcriptomics has been widely applied to investigate the mechanisms underlying metamorphosis in invertebrates, revealing the critical pathways and genes that regulate metamorphosis and providing novel insights (Shikuma et al., 2016; He et al., 2021). Herein, to further investigate the mechanism by which juvenile oysters induce the metamorphosis of R. venosa larvae, we obtained the mRNA expression profiles of competent larvae exposed to juvenile oysters for 2 hours (M2) and 12 hours (M12) and before exposure to juvenile oysters (control, Con) via sequencing on an Illumina NovaSeq 6000 platform. Furthermore, we identified the differentially expressed mRNAs (differentially expressed genes, DEGs) and performed trend analysis of gene expression based on their transcripts per million (TPM) values to further screen out the genes that were consistently regulated during exposure to juvenile oysters. Then, these genes were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The findings of this study will enhance understanding of the metamorphosis mechanism of R. venosa, which may not only aid in the development of artificial breeding and culturing but also reveal the invasion mechanism and support the protection of wild populations of oysters and other bivalves.

The food or prey of some animals not only provides them with the necessary nutrients for growth and development but also plays an important role in their development at a critical time. One of the most dramatic examples is when the food or prey induces animal metamorphosis (Morse et al., 1979; Rodriguez et al., 1993; Taris et al., 2010). Although representative species from diverse taxa undergo food-induced metamorphosis, little is known about the processes that occur within the animals as they detect the cues produced by their food. To further understand the mechanism by which juvenile oysters induce the metamorphosis of R. venosa larvae, we performed RNA-seq and analyzed changes in gene expression at different time points upon exposure to metamorphosis cues, the juvenile oysters. Then, we conducted functional enrichment for these genes and identified the critical signaling pathways in the process. The present results may provide new insights into the regulatory mechanisms of metamorphosis in marine invertebrates.

In the present study, GO enrichment analysis revealed that 189 DEGs that responded early to the presence of juvenile oysters were significantly enriched with two GO terms: DNA-binding transcription factor activity and transcription regulator. These genes included some critical transcription factor genes, such as Maf, AP-1, MafK, MafAa, and transforming protein v-Fos/v-Fox, and most of the genes were upregulated. A previous study has also indicated that these genes are Jun or Fos genes (transcription factor AP-1 gene family), which are prototypical immediate early genes. Immediate early genes are characterized by a rapid and transient activation of transcription in response to changes of environmental conditions, such as growth factors, cytokines, tumor promoters, carcinogens, and expression of certain oncogenes (Angel and Hess, 2010). The transcription factor AP-1 gene family participates in many pathways, such as the MAPK signaling pathway, Wnt signaling pathway and apoptosis, which have been proven to be important in the metamorphoses of marine invertebrate animals (Angel and Hess, 2010). Therefore, we speculate that the transcription factor AP-1 may be a critical initiator in competent larvae of R. venosa in response to the metamorphosis cues of juvenile oysters that regulates other subsequent processes.

A previous study has indicated that the most critical members of the class of protein kinases regulating the activity of AP-1 in response to extracellular stimuli are MAPKs (Angel and Hess, 2010). In addition, researchers have shown that the MAPK signaling pathway is induced by the metamorphosis cue in tubeworm Hydroides elegans and regulates the downstream process of metamorphosis (Shikuma et al., 2016; Wang and Qian, 2010), which also occurs in other marine invertebrate animals, such as corals, ascidians and barnacles (Chambon et al., 2007; He et al., 2012; Siboni et al., 2014). Interestingly, we also found that the MAPK signaling pathway was significantly enriched in the gene set of profile 2 in the trend analysis, which may mean that in competent larvae of R. venosa, MAPK signaling participates in the response to metamorphosis cues.

Chambon et al. (2007) found that the MAPK signaling pathway regulates gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles, and apoptosis occurs during metamorphosis in almost all invertebrates, including R. venosa (Song et al., 2016a). In the present study, we found that the expression of IAPs was significantly increased upon exposure to juvenile oysters. IAP expression is also upregulated after metamorphosis in juveniles of R. venosa (Shi, 2022). IAPs are important regulators that participate in the sophisticated molecular machineries in cells that guard against inappropriate or premature apoptosis to counter various stimuli capable of triggering death (Song et al., 2021). IAPs are also highly upregulated in response to various environmental stresses, including heat, air exposure, and low-salinity stress, in some marine invertebrate animals (Hu et al., 2022). The presence of oysters can also be considered an environmental factor for competent R. venosa larvae, and the upregulation of IAPs in the present results indicates that apoptosis is tightly regulated during the metamorphosis of R. venosa. The observed upregulation also suggests that apoptosis is induced by the presence of oysters during the metamorphosis of R. venosa. Furthermore, we found that the transcription factor AP-1 affected the expression of some proapoptotic genes, including p53, Fas, Fas-L, Bim and HRK, which supports the findings of a previous study showing that AP-1 target genes are involved in the regulation of apoptosis (Angel and Hess, 2010).

Past studies have revealed that, as a molecular chaperone of nitric oxide synthase (NOS), HSP90 is also a negative regulator (repressor) of metamorphosis in a diverse range of marine invertebrates (Bishop and Brandhorst, 2001; Bishop et al., 2001; Hens et al., 2006; Taris et al., 2009). However, Ueda and Degnan (2014) had a different discovery: that inhibition of NOS reduces rates of metamorphosis, which means that NO facilitates, rather than represses, induction of metamorphosis in Haliotis asinina. Additionally, Ueda and Degnan (2014) tested the hypothesis of NOS-HSP90 interaction at the initiation of settlement and metamorphosis because they found that the spatial and temporal expression trends of NOS and HSP90 were not consistent. In the present study, we found that HSP90 genes were significantly downregulated after exposure to oysters for 12 hours ( Figure 4C ), which might be considered to be a signal of metamorphosis induced by the oysters, but the specific function of HSP90 needs further investigation.

In addition to HSP90 genes, several HSP70 genes were also significantly downregulated in oysters ( Figure 4C ). Similar trends of downregulation were observed in a study on gene expression during the metamorphosis of R. venosa (Shi et al., 2022). HSP70 first received attention for its role in the response to heat shock (Welch, 1993) and has also been found to be critical for the folding and assembly of other cellular proteins (Ritossa, 1962). HSP70 genes also participate in the regulation of the MAPK signaling pathway (Patel, 2009), which has been widely reported to participate in the regulation of metamorphosis in several marine invertebrate animals mentioned above. Porto et al. (2018) reported that HSP70 can act as a signaling molecule, modulating MAPK downstream signaling during memory consolidation in the hippocampus. Patel (2009) also indicated that HSP70 plays a role in the induction of p38MAPK in animals to respond differently to high osmotic stress. Therefore, we speculate that HSP70 may also participate in the MAPK signaling pathway and be related to the metamorphosis of R. venosa; however, the specific effect of HSP70 on metamorphosis needs further investigation, especially since no previous studies have reported the relationship between HSP70 and metamorphosis in marine invertebrates.

The Wnt signaling pathway, which has been widely studied, regulates cell proliferation, migration, differentiation and survival during animal development as well as the maintenance of homeostasis and regeneration (Liu et al., 2008; De Robertis, 2010; Clevers and Nusse, 2012). In the trend analysis, we found that the Wnt signaling pathway was significantly enriched in profile 2, a gene set that was decreased at the early stage during exposure to juvenile oysters. This finding may suggest that the Wnt signaling pathway participates in the metamorphosis of R. venosa. Chandramouli et al. (2013) also found Wnt signaling pathway enrichment during the larval metamorphosis of the polychaete worm Pseudopolydora vexillosa in transcriptome studies. Furthermore, Yue et al. (2012) have reported that Wnt signaling pathways may be involved in the metamorphosis of the bryozoan Bugula neritina. However, the specific function of the Wnt signaling pathway in the metamorphosis of marine invertebrates needs further investigation.

In this study, the gene set responses to metamorphosis cues (juvenile oysters) in R. venosa (I-DEGs and F-DEGs) were identified, and GO and KEGG enrichment analyses were further performed on these gene sets. We found that DNA-binding transcription factor activity and transcription regulator activity were enriched for the I-DEGs, such as the transcription factor AP-1, which is a prototypical immediate early gene that rapidly and transiently activates transcription in response to changes in environmental conditions. The molecular chaperone of NOS, HSP90, exhibited lower expression in the M12 group than in the Con group, while the expression of IAPs was significantly increased upon exposure to juvenile oysters. Additionally, the Wnt signaling pathway and MAPK signaling pathway were enriched in the trend analysis. These pathways may also play critical regulatory roles in the response to juvenile oysters. Taken together, the results show that competent larvae rapidly respond to the presence of oysters by expressing some immediate early genes, such as the transcription factor AP-1. These genes may further regulate downstream pathways such as the MAPK signaling pathway and result in subsequent changes, including a decrease in HSP90 and an increase in IAPs. These changes together may regulate the metamorphosis of the larvae.

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 .

The studies involving animals were reviewed and approved by the Animal Welfare Committee of the Institute of Oceanology, Chinese Academy of Sciences.

TZ and HS conceived and designed the experiments. M-JY and HS conducted the experiments. M-JY analyzed the data. YS, Z-SL, PS, ZH, CZ, P-PH and Z-LY contributed reagents, materials, and analytical tools. M-JY wrote the manuscript. All authors contributed to the article and approved the submitted version.