Chinese tongue sole (Cynoglossus semilaevis) is an indigenous flatfish species with high economic value in China. In the larvae stage, flatfishes undergo a morphological metamorphosis toward an asymmetric body plan (both eyes on the same side) to adapt the benthic dwelling lifestyle (Chen et al., 2014; Fox et al., 2018; Lü et al., 2021). Then, the body color of ocular and blind side exhibited as black-brown and pure white respectively in the wild. However, under commercial production environments, the coat color of the blind side is prone to pigment staining, which is referred to as blind-side hypermelanosis. The high proportion of hypermelanic individuals in production populations combined with their high area ratio of hypermelanosis has emerged as a major concern for fish farmers and consumers, as this yields an inferior product and substantially lower profit (Li et al., 2021a; Li et al., 2021b). Numerous key genes associated with blind-side hypermelanosis phenotype have been screened and identified, and single-nucleotide polymorphism mutations were detected in transcriptional level in flatfishes (Peng et al., 2020; Li et al., 2021a; Zhang et al., 2021). However, there is no clear evidence explaining the underlying regulatory mechanism of blind-side hypermelanosis especially regarding the regulation of non-coding RNA (ncRNA).

Evidence gathered over the past decade suggests that circular RNAs (circRNAs) as endogenous ncRNAs play crucial roles in various biological processes in eukaryotic cells (Memczak et al., 2013; Ebbesen et al., 2017). However, the functional mechanisms of circRNA are both complex and controversial. For a better understanding of the functions of circRNAs, firstly, the functions of their host genes should be focused. For example, circRNA can bind to its host gene locus through R-loop (RNA : DNA hybrid) formation, resulting in transcriptional pausing, which leads to phenotypic changes (Conn et al., 2017; Xu et al., 2020). Secondly, circRNAs could function as microRNA (miRNA) molecule sponges to counteract the inhibition of miRNA-mediated messenger RNA (mRNA) through competing endogenous RNA (ceRNA) networks (Salmena et al., 2011). In humans, recent studies detected thousands of circRNAs in melanocytes that were found to play key roles in cell proliferation and invasion (Wang et al., 2018b). Furthermore, circRNA can relieve the inhibition of miRNA on the tyr gene to promote melanogenesis through a ceRNA regulation network (Jiang et al., 2020). However, the specific roles and mechanisms of circRNAs in melanogenesis remain poorly understood, and further studies are needed (Zhou et al., 2021). Currently, pigmentation-related miRNAs have been identified in several fish species, including red tilapia (Oreochromis sp. red tilapia) (Wang et al., 2018a), common carp (Cyprinus carpio) (Yan et al., 2013), and Koi carp (Cyprinus carpio carpio) (Luo et al., 2018; Dong et al., 2020). Literature focusing on circRNA and ceRNA regulatory network involved in pigmentation is not available in fishes.

Therefore, in the current study, high-throughput sequencing and bioinformatics analyses were performed to decode the potential roles of circRNA in tongue sole blind-side hypermelanosis. Host genes of circRNAs were identified and functionally annotated. For the first time, the circRNA-miRNA-mRNA regulatory network associated with the blind-side hypermelanosis trait was predicted. This research expands the spectrum of non-coding regulatory mechanisms underpinning malpigmentation in flatfish.

Despite the contribution of genetic and plastic traits to environmental adaptation, phenotypic plasticity allows species to survive in a wider range of conditions and respond quicker to environmental changes (Agrawal, 2001). One significant example of phenotypic plasticity in animals is body color change. In flatfish, blind-side pigment distribution abnormalities are often observed in farming populations. To understand the processes responsible for this adaptation and evolution, research linking to genetic factor and phenotype of body color is necessary (Chen et al., 2019). Recently, an increasing number of studies have focused on circRNAs because of their involvement in pigmentation regulatory function (Zhu et al., 2020). However, research on circRNAs in non-model organisms such as flatfish species is still lacking, especially studies on melanogenesis mechanism.

In the present paper, an overview of circRNAs in the blind-side skins of tongue sole is provided. A total of 73 DEcircRNAs were found between BN and BH. GO enrichment and KEGG pathway analyses for host genes of these DEcircRNAs showed that the variation in hypermelanosis was related to cellular components, such as “Golgi membrane”, “Golgi apparatus part”, and “intrinsic component of Golgi membrane”. Tyrosinase family proteins (e.g., TYR and TYRP1) play important roles in the melanin biosynthetic pathway. These two enzymes can be trafficked through the Golgi apparatus and then join type II melanosomes, thus promoting the deposition of melanin that finally develops into a mature melanosome (Dooley et al., 2012). On this basis, it can be speculated that circRNAs, such as novel_circ_000033, novel_circ_000485, and novel_circ_000748, might regulate their host genes to affect TYR and TYRP1 transport on the Golgi. Additionally, the host gene (Slc8a2) of novel_circ_000935 was significantly enriched in “calcium ion export” and “cGMP-PKG signaling pathway”. Slc8a2 is a member of the solute carrier 8 family and encodes a Na⁺/Ca²⁺ exchanger to mediate cellular Ca²⁺ efflux/influx (Quednau et al., 2004). The cGMP-PKG signaling pathway can inhibit Ca²⁺ currents (Sandoval et al., 2017). Increasing intracellular Ca²⁺ can mediate pigment aggregation (Aspengren et al., 2003). Therefore, the downregulated novel_circ_000935 and Slc8a2 may partially explain the blind-side hypermelanosis in tongue sole.

Understanding concerning details on the body color change depends on knowledge of where pigment translocations originate on isolated melanophores from fish (Sköld et al., 2016). The aggregation of pigment in fish is regulated by hormonal stimuli or direct innervation (Aspengren et al., 2003). When melanogenesis is activated, molecular motors carry melanosomes along the cytoskeleton, dispersing them throughout the cell or aggregating them in the cell center (Aspengren et al., 2009). During melanogenesis, pigment translocation depends on actin filaments and microtubules. In this study, it was found that circRNAs (novel_circ_000176, novel_circ_000290, novel_circ_000737, novel_circ_000839, novel_circ_000846, novel_circ_001038, novel_circ_001147, novel_circ_001237, and novel_circ_001416) mediated in the ceRNA network were associated with stimulus/perception-related biological processes. Examples are “sensory perception of mechanical stimulus”, “sensory perception”, and “neurological system process”. The relevant genes (e.g., slc17a8, Espn, MYO15A, and PJVK) mined in this study have been suggested to play important roles in mediating sensory transduction in various mechanosensory and chemosensory processes (Sekerkova et al., 2008; Zhang et al., 2015; Ryu et al., 2017; Zhu et al., 2018; Zhang et al., 2019). Empirically, blind-side skin of tongue sole would show varying degrees of abrasions ( Figure 7A ) because of long-term captivity in aquaculture tanks (that are relatively smooth). A plausible explanation for this is that to better adapt to the benthic life, sensory perception-related genes are downregulated, and tongue sole fish may have “tired” of the smooth environment.

Coincidentally, blind-side hypermelanosis is accompanied by a morphological change of scales in the corresponding parts, where the scales cannot develop into cycloid scales ( Figure 7B ) but remain ctenoid scales ( Figure 7C ). Normally, after metamorphosis in the larva stage, the blind side is initially covered by ctenoid scales (without pigmentation); then, ctenoid scales gradually change into cycloid scales in the juvenile stage. While, after metamorphosis, the ocular side is covered by ctenoid scales throughout the later life stages. This demonstrates that the morphological structure of scales may be closely associated with hypermelanosis. Fish scales originate from the dermis of the skin (Sire and Akimenko, 2004). In nature, flatfish bury themselves in sediment such as sand for camouflage, predation, and protection. To avoid removal of sand from the ocular-side skin by water currents, ctenoid scales lead to an increase of friction forces (Spinner et al., 2016). From this perspective, the interruption of morphological change of scales on the blind side may imply that tongue sole fish could tackle the relatively smooth artificial environment by maintaining ctenoid scales on the blind side. In this study, it was found that certain host genes of circRNAs were significantly enriched in “Wnt signaling pathway” and “tight junction” pathway. Previous studies reported that these two pathways are related to the development of fish scales (Aman et al., 2018; Wang et al., 2021). However, the causal relationship between ctenoid scale maintenance and hypermelanosis is still unclear.

In fishes, morphological color change requires weeks or months, which implies that substantial increases or decreases in the numbers of chromatophores are needed (Sköld et al., 2016). A previous report regarding tongue sole has indicated that the melanocyte is a requisite for blind-side pigmentation (Shi et al., 2015). Melanin is enriched in melanocytes and mainly synthesized from tyrosine. Tyrosine aminotransferase (TAT) can catalyze phenylalanine into tyrosine (Mehere et al., 2010). Increased or reduced expression of TAT affects the synthesis of tyrosine and melanin. In the present study, it was predicted that novel_circ_000405 may act as a sponge for miR-1193-3p and miR-495-3p to regulate TAT expression. The expression level of TAT in the BH group was significantly lower than that in the BN group. This suggests that novel_circ_000405 may also play an important role in blind-side hypermelanosis in tongue sole.

This is the first paper to describe the comprehensive expression of circRNAs of the blind-side hypermelanosis in tongue sole and predict the functional profile of circRNAs based on host gene analysis and ceRNA regulatory network construction. Numerous key circRNAs of functional significance were identified. The results suggest that circRNAs may play vital roles in blind-side pigmentation, and valuable insights into the molecular mechanism of hypermelanosis in flatfish are provided. It is concluded that blind-side hypermelanosis is an interesting game between fish and aquaculture environment. The findings also reveal adaptive trait evolution to the aquaculture environment.

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 below: NCBI [accession: PRJNA760350].

The present study was approved by the Institutional Animal Care and Use Ethics Committee of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China. When sampling, fishes were anesthetized with MS-222 to minimize suffering.

YH Methodology, software, and writing—original draft preparation. YL Conceptualization, methodology, data curation, visualization, validation, and writing—reviewing and editing. PC Visualization and investigation. SC Supervision and writing—reviewing and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the China Agriculture Research System of MOF and MARA (CARS-47-G03), National Natural Science Foundation of China (31702333), the Innovative Team Project of Chinese Academy of Fishery Sciences (2020TD20), and Taishan Scholar Climbing Program of Shandong Province China.

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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