Transcriptome analysis and candidate gene identification reveals insights into the molecular mechanisms of hypermelanosis in Chinese tongue sole(Cynoglossus semilaevis)

Yngzhen Li, Peng Cheng, Ming Li, Yunri Hu, Zhongki Cui,b, Chunto Zhng,Songlin Chen,b,c

aKey Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Qingdao, 266071, China

bLaboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, 266237,China

cNational Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China

Keywords:

Cynoglossus semilaevis

Hypermelanosis

Transcriptome

Flatfish

A B S T R A C T

Blind-side hypermelanosis has emerged as a major concern in flatfish aquaculture worldwide, including tongue sole (Cynoglossus semilaevis) in China.The causative gene and the molecular basis are still unclear.In this study,comparative transcriptome analyses were performed using different skin tissues of tongue sole: ocular-side normal (pigmented) skin, blind-side normal (non-pigmented) skin and blind-side hypermelanotic (pigmented)skin.Finally, 60 key hypermelanosis-related genes were mined, providing potential candidate gene resources involved in blind-side hypermelanosis.These genes were selected based on the log2(FoldChange) and false discovery rate (FDR) values (with corresponding P-Values ＜0.05), and they were verified in other species to assess if they were directly or indirectly related to melanogenesis.The protein-protein interaction network of these 60 genes and the relationship between tyr and other key hypermelanosis-related genes were illustrated.The qRT-PCR validation of 16 differentially expressed genes (DEGs) showed that the data of qRT-PCR were consistent with those of RNA-seq.Further analyses revealed that the selected DEGs were significantly overrepresented in several pigment metabolic processes and in the melanogenesis pathway.Our results may imply that blind-side hypermelanosis is a pattern of environmental regulation of gene expression and adaptation in flatfish.Membrane transport proteins (such as OCA2 and SLC45A2) may serve as a “switch” for melanogenesis in tongue sole.Overall, this study provided novel insights into the molecular mechanism of hypermelanosis in flatfish species and will facilitate future selective breeding of tongue sole for this market-favoured trait in aquaculture.

1.Introduction

Flatfish is an important economic species which is cultured worldwide, including East Asia, Europe and North America.All the flatfish species undergo metamorphosis during early development, before which they are bilaterally symmetrical and after which they are bilaterally asymmetrical with both eyes on the same body side (ocular side).The skin of the ocular side becomes pigmented (black-brown) and on the blind side is non-pigmented (pure white) (Kang, Kim, & Chang, 2011).However, in flatfish aquaculture (i.e., under hatchery rearing conditions), staining-type hypermelanosis on the blind side after metamorphosis has emerged as a serious problem for production of Japanese(olive) flounder (Paralichthys olivaceus) and tongue sole (Cynoglossus semilaevis) in east Asian counties.In Japanese flounder, the pigmentation of the blind side starts from the caudal peduncle area, and spreads across the entire body on the blind side of the fish and has a distinctive appearance (Isojima, Makino, Takakusagi, & Tagawa, 2013; Kang &Kim, 2012a).While, in tongue sole, it usually develops from randomly generated small speckles and then spreads across the whole body of the blind side.The normal and hypermelanotic body colors are shown in Fig.1.

Fig.1.Normal body color and hypermelanosis in Chinese tongue sole.A:normal pigmentation on the ocular side; B: normal (none) pigmentation on the blind side; C: hypermelanosis on the blind side.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In flatfish aquaculture, for example Japanese flounder in Japan and Korea (Kang & Kim, 2012a, 2012b; Nakata et al., 2017) and tongue sole in China, a considerable proportion of individuals express blind-side hypermelanosis, depreciating the market value of the fish as it transmits a negative perception of the product to consumers.Pigmentation is a complex trait that depends on genetics and other environmental factors.Laboratory and field studies have attempted to determine the causes of hypermelanosis and some effective prevention techniques have been developed, for example roughing the bottom of rearing tanks with sand or other substrates, adjusting light, delaying the time of feeding artificial diet,fine-tuning stocking density and background color (Iwata &Kikuchi, 1998; Amiya et al., 2005; Isojima, Tsuji, Masuda, & Tagawa,2013; Yamanome, Amano, & Takahashi, 2005; Yamanome, Amano,Amiya, & Takahashi, 2007; Yamanome, Chiba, & Takahashi, 2007; Kang et al., 2011; Kang & Kim, 2012b, 2013; Nakata et al., 2017).Moreover,studies from an endocrinological perspective revealed the possible mechanisms of hypermelanosis (Amiya et al., 2005; Yamanome et al.,2005; Yamanome, Chiba, & Takahashi, 2007; Yamada, Donai, Okauchi,Tagawa, & Araki, 2011; Kang & Kim, 2012b; Yoshikawa, Matsuda,Takahashi, & Tagawa, 2013; Matsuda, Yamamoto, Masuda, & Tagawa,2018).However, for flatfish species, the molecular mechanisms of hypermelanosis are still unclear.Recent studies in Japanese flounder revealed that genes involved in thyroid synthesis and the melanogenesis pathways play an important role in blind side hypermelanosis (Peng et al., 2020).

Melanin has been verified as one of the most important pigments for maintaining hypermelanic coat color and it widely exists in vertebrates,including fishes, reptiles, birds, mammals and humans (Hoekstra, 2006).Melanin synthesis is catalyzed by tyrosinase which is the fundamental substrate in this process (D’Mello, Finlay, Baguley, & Askarian-Amiri,2016).Mutation of the key genes encoding tyrosinase or other enzymes in the melanogenesis pathway may result in albinism, which is another phenotype of malpigmentation.Mutated genes associated with albinism, such astyr,oca2,tyrp1,slc45a2andhsp4, have been identified in numerous types of vertebrates, including fishes, mice, birds and humans.For fish body color, previous studies mainly focused on albinism, and combined with studies in other vertebrates, the underlying molecular mechanisms of albinism are regarded to be gene mutations.From this perspective, we speculate that the molecular mechanism of blind-side hypermelanosis in flatfish seems very different from albinism.It seems probable that some environmental factors activate key genes which should be suppressed in the blind-side skins.

Chinese tongue sole in a commercially important indigenous marine flatfish which is widely cultured and it is one of the most expensive farmed fish species on the market in China (Guan et al., 2018; Li et al.,2019; Song et al., 2020).The complete genome of the Chinese tongue sole was published in 2014 (Chen et al., 2014), and it has provided a foundation for tongue sole RNA sequencing (RNA-Seq) studies.RNA-Seq technology has been widely used in functional genomics research in fish.With the rapid development of next-generation sequencing technologies, RNA-Seq as an important tool is often used to reveal the molecular mechanism of specific physiological processes.

In this study, we sequenced the skin transcriptome of the blind-side hypermelanotic skin, blind-side normal skin and ocular-side normal skin from hypermelanotic tongue sole individuals using Illumina sequencing technology.The major objective of this study was to investigate hypermelanosis-related differentially expressed genes (DEGs) and to propose a hypothesis that blind-side hypermelanosis is an environmental regulation of gene expression and adaptation in flatfish.Further,the tremendous amount of data obtained from RNA-Seq provide fundamental information and candidate genes for the genetic improvement of tongue sole.

2.Materials and methods

2.1.Fish material and sample collection

Juvenile tongue sole fish used for sampling were from the same fullsib family which were collected from our flatfish breeding center in Tangshan, China.No hypermelanosis were observed visually on the blind side for the offspring of this family untill 60 days post hatch (dph).While at 80 dph, about 50% (randomly sampled 50 individuals three times for statistics) individuals exhibited hypermelanosis on the blind side.Three blind-side hypermelanotic fish (total length 8-10 cm) with about 50% pigmented area were randomly selected to collect skin tissues (each approximate 0.5 cm2).Then three-group samples (each with three biological replicates) were obtained, i.e., ocular-side normal(pigmented) skin (ON), blind-side normal (non-pigmented) skin (BN)and blind-side hypermelanotic (pigmented) skin (BH).Before sampling,fish were anesthetized with MS-222 (Maya Reagent, Zhejiang, China).All the tissues were immediately frozen and stored in liquid nitrogen until use.

2.2.RNA extraction, library construction and sequencing

Total RNA of all the skin tissue samples (N=9) was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.A NanoDrop 2000 (Termo Scientific, Wilmington, DE, USA) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA) were used to assess the quantity and quality of total RNA.Then a total of nine cDNA libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA,USA) according to the manufacturer’s instructions.The cDNA fragments were purified, end repaired, poly(A) added, and ligated to Illumina sequencing adapters.The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq 2500 by Gene Denovo Biotechnology Co., Ltd.(Guangzhou, China).

2.3.Alignment with tongue sole reference genome

The raw reads and low-quality bases (Q ≤20) were filtered by fastp(Chen, Zhang, Chen, & Gu, 2018) to obtain clean reads.Clean reads were mapped to the tongue sole reference genome (https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_other/Cynoglossus_semilaevis/latest_assembly_versions/GCF_000523025.1_Cse_v1.0/) using HISAT2.2.4 (Kim Langmead, & Salzberg, 2015).The mapped reads of each sample were assembled using StringTie v1.3.1 (Pertea et al., 2015; Pertea, Kim,Pertea, Leek, & Salzberg, 2016) in a reference-based approach.

2.4.Identification of differentially expressed genes (DEGs)

The gene expression levels were calculated based on fragments per kilobase of transcripts per million fragments mapped (FPKM) values using RNA-Seq by Expectation Maximization (RSEM) with default setting (Li & Dewey, 2011).The DEseq2 software was used to detect DEGs between different groups (Love, Huber, & Anders, 2014).The genes with the parameters of false discovery rate (FDR)＜0.05 and |log2FoldChange| ≥1 were assigned as DEGs.

2.5.Enrichment analysis and protein-protein interaction

Gene ontology (GO) enrichment analyses of DEGs was performed by the GOseq R package and gene length bias was corrected (Young,Wake field, Smyth, & Oshlack, 2010).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to identify significantly enriched metabolic pathways.After hypergeometric test, GO terms and KEGG pathways with p＜0.05 were considered significantly enriched.Protein-protein interaction networks were identified using String v10 (Szklarczyk et al., 2015), which determined genes as nodes and interactions as lines in a network.The network file was visualized using Cytoscape (v3.7.1) software to present key gene biological interactions (Merico, Gfeller, & Bader, 2009; Shannon et al.,2003).

2.6.Quantitative real-time PCR validation

To validate the RNA-Seq results, 16 DEGs were selected for quantitative real-time PCR (qRT-PCR) analyses.The selection criteria of these genes were mainly based on the log2(FoldChange) and FDR values with corresponding P-Values＜0.05, and they were verified in other species to confirm they were directly or indirectly related to melanogenesis.Primers were designed based on the coding sequence of identified genes from the tongue sole genome (Supplementary Table 1).The β-actin gene was used as an internal reference.The cDNA was synthesized from 1 μg of total RNA for each sample.PCR amplification experiments were performed in triplicates.The thermal cycle for SYBR Green RT-PCR was 95 ℃ for 90s, followed by 40 cycles of 95 ℃ for 5s and 60 ℃ for 15s, and 72 ℃ for 20s.The expression level of target genes relative to β-actin was normalized by the 2-ΔΔCTmethod (Livak &Schmittgen, 2001).The fold change of 16 genes in all comparison groups obtained by RNA-Seq were calculated by FPKM.These genes’ log10 fold change values of qRT-PCR and RNA-seq were used for graphical presentation.

2.7.Statistical analysis

IBM SPSS Amos 21.0 software was used to analyze the data, which were presented as the mean ±standard error (SE), and one-way analysis of variance (ANOVA) was used to determine the significance of differences between different groups.The level of statistical significance was set at p＜0.05.

3.Results

3.1.Summary and assessment of RNA-Seq data

In the present study, to better understand the blind side hypermelanotic mechanisms of tongue sole, a total of 9 samples from ocular and blind sides of hypermelanotic fish were used for Illumina-based RNA sequencing.As a result, a mean of 41.93 million filtered clean reads(～99.8% from raw reads) and a mean of 40.98 million rRNA-filtered reads (～97.7% from clean reads) was obtained.The mapped clean reads percentage of the 9 cDNA libraries ranged from 91.78% to 93.16%.The means of Q20 and GC percentage for all the libraries were 97.24% and 52.50% respectively (Supplementary Table 2), which confirmed the high quality of the RNA-Seq data.Raw sequencing data has been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA662432.

3.2.Identification of DEGs

To identify DEGs involved in hypermelanosis, the analyses were mainly focused on the difference between BN and BH groups.While the comparative analyses between BN and ON, ON and BH were also performed as a reference.Genes with FDR＜0.05 and |log2FoldChange| ≥ 1 were classified as DEGs.The total and shared numbers of DEGs between different comparative groups, and up-regulated and down-regulated numbers of DEGs within each comparative group were shown in Fig.2.In BN-vs-BH group, the (50 DEGs) excluded from the other two comparative groups were expected to be highly relevant for hypermelanosis (hypermelanosis specific) and those overlapping genes (85 DEGs) excluded from ON-vs-BH group were expected to be highly relevant to the maintenance of pigmentation.

Fig.2.Venn diagram of differentially expressed genes (DEGs) and the number of up- and down-regulated DEGs in three comparative groups.BN: blind-side normal skin; BH: blind-side hypermelanotic skin; ON: ocular-side normal skin.

3.3.GO and KEGG functional enrichment analysis of DEGs

To further understand the function of DEGs, GO and KEGG enrichment analyses were performed to investigate the molecular mechanisms of hypermelanosis or pigmentation trait modules.Data from the three groups were pairwise compared, i.e., BN-vs-BH, BN-vs-ON and ON-vs-BH.GO term enrichment analysis results of the three comparative groups are shown in Fig.3 and Supplementary Figure 1 (more inferior level GO terms in biological process).All the DEGs in each comparative group were classified into three ontic categories: biological process,molecular function and cellular component and involved 17, 8 and 13 subcategories respectively (Fig.3).From the top 20 significantly enriched GO terms in the biological process category, 25, 16 and 6 DEGs were obtained in BN-vs-BH, BN-vs-ON and ON-vs-BH groups respectively, where 13 GO terms, such as melanin metabolic process, secondary metabolic process and pigment metabolic process, were shared between BN-vs-BH and BN-vs-ON, while there was only one term shared between BN-vs-BH and ON-vs-BH, and no terms shared between BN-vs-ON and ON-vs-BH (Supplementary Figure 1).The shared DEGs from the top 20 significantly enriched GO terms in the biological process category are shown in Fig.4A.Complete GO enrichment analysis results are shown in Supplementary Table 3.These results revealed that the mechanisms of blind-side hypermelanosis are similar to ocular-side pigmentation to a large extent.

Fig.3.Gene ontology (GO) functional classification of differentially expressed genes (DEGs) in the three compared groups.BN: blind-side normal skin; BH: blind-side hypermelanotic skin; ON: ocular-side normal skin.

Fig.4.The total and shared numbers of differentially expressed genes (DEGs) from the top 20 significantly enriched gene ontology (GO) terms in the biological process category (A) and the total and shared numbers of DEGs from all significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (B) in/among different compared groups.BN: blind-side normal skin; BH: blind-side hypermelanotic skin; ON: ocular-side normal skin; BP: biological process category.

The KEGG pathway enrichment analysis results of the three groups are shown in Fig.5 (top 20 pathways).In BN-vs-BH, BN-vs-ON and ON-vs-BH group, 62, 60 and 38 DEGs were assigned to 101, 119 and 111 pathways respectively, of which 13, 14 and 20 pathways were signi ficantly enriched, respectively (P-value＜0.05).Pathways, such as tyrosine metabolism and melanogenesis, were key pathways highly related to blind-side hypermelanosis and/or pigmentation.In these significantly enriched pathways, the numbers of DEGs between different groups and the shared DEGs among different groups are shown in Fig.4B.Complete KEGG enrichment analysis results are shown in Supplementary Table 4.

Fig.5.Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment bar chart of differentially expressed genes (DEGs) in top 20 pathways in different comparative groups.BN: blind-side normal skin; BH: blind-side hypermelanotic skin; ON: ocular-side normal skin.

3.4.Key hypermelanosis-related DEGs and protein-protein interaction networks

In this study, we mainly focused on mining DEGs related to hypermelanosis, though in most cases hypermelanosis-related DEGs were also highly relevant to ocular-side pigmentation.Based on the transcriptions and GO and KEGG functional enrichment analyses, 60 key hypermelanosis-related DEGs (43 up- and 17 down-regulated genes)were selected for further investigation.The detailed information about these genes is listed in Supplementary Table 5.Among these genes, 16 DEGs were significant and will be targeted in future studies.

To reveal the relationship of the hypermelanosis-related DEGs,protein-protein interaction network analysis was conducted (Fig.6).Most of the genes had strong expression correlations, some nodes with many edges were assumed to be hub genes, such astyr,tyrp1, etc, which played significant roles in melanogenesis and/or pigmentation maintenance.The relationship between TYR and the other 59 genes (55 proteins) are presented in Fig.7.

Fig.6.Protein-protein interaction network of hypermelanosis-related differentially expressed genes (DEGs).Gene symbol with red and green highlight indicates upand down-regulation, respectively.Solid lines and dotted lines indicate positive and negative correlations respectively, and the thickness of the lines indicates the degree of correlation, more thick more relevant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig.7.Relationship between TYR and other selected hypermelanosis-related differentially expressed genes(DEGs).Gene symbol with red and green highlight indicates up- and down-regulation respectively.Solid lines and dotted lines indicate direct and indirect correlation respectively, and the thickness of lines indicates the degree of correlation, more thick more relevant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5.Validation of differentially expressed genes by qRT- PCR

To validate the expression profiles, the relative mRNA expression levels of 16 selected DEGs were measured using qRT-PCR.Results showed that the data of qRT-PCR were consistent with those of RNA-seq(Fig.8), confirming the reliability of the data obtained by RNA-seq.The relative expression in blind-side hypermelanotic (BH), blind-side normal(BN) and ocular-side normal (ON) skin tissues were shown in Supplementary Figure 2.

Fig.8.Correlation between qRT-PCR and RNA sequencing for the 16 selected key genes.BN: blind-side normal skin; BH: blind-side hypermelanotic skin; ON: ocularside normal skin.

4.Discussion

4.1.Malpigmentation in tongue sole: an empirical summary and implication on selective breeding

In flatfish, pigment abnormalities are often observed, especially in hatcheries under aquaculture environments.The abnormal body color can be mainly classified into two types, i.e., ocular-side pseudo-albinism(ocular-side skin total or partial albinism but eyes pigmented) and blindside hypermelanosis (Venizelos & Benetti, 1999).In tongue sole,empirically, pseudo-albinism usually occurs with metamorphosis,associated sometimes with impaired eye migration, i.e., bilateral eyes(each side with one eye) or smaller interorbital space, either-or of the two cases almost 100% occurred when skin around eye(s) was albinistic,and we firmly believe that the cranial bone is remodeled at the same time.Clues and evidence can be found and confirmed from studies in another flatfish, Senegalese sole (Boglino et al., 2014).In tongue sloe,when metamorphosis was achieved, the pattern of albinism on ocular side was permanent, which means the area ratio was constant throughout grow-out period with no spread or reduction.While for hypermelanosis in tongue sole, we believe that there is a process of accumulation after metamorphosis, till it can be observed by eyes.And in most cases, hypermelanosis spreads during the grow-out period with no reduction under artificial aquaculture environments.Abnormally pigmented tongue sole and other flatfish are regarded to have inferior quality and this lowers their market value considerably.

In aquaculture populations, blind-side hypermelanosis is variable in frequency, with a wide range of individual hypermelanosis ratio,ranging from 10%-90% in most cases, and differing between hatcheries or farms.And among hypermelanotic individuals, the pigmented area ratio ranges from 0 to nearly 100%.Given these facts, blind-side hypermelanosis can be regard as a quantitative (continuous) trait,which is controlled by some genes (minor genes), and the effects of these minor genes are generally additive.According to our experiences and previous studies, hypermelanosis trait is very sensitive to environmental factors.Thus, it is expected that non-hypermelanosis trait can be genetically improved by selective breeding based on methodologies in quantitative genetics.

4.2.Possible molecular mechanisms of hypermelanosis in tongue sole

Many studies from an environmental control and nutritional perspective have been conducted to prevent or lower the incidence of blind-side hypermelanosis in flatfish.However, studies on molecular mechanisms of hypermelanosis in flatfish especially in tongue sole are still scarce.In this study, for the first time, comparative transcriptomic analysis by using data from different pigmented skin tissues was performed to investigate the underlying mechanisms of hypermelanosis.The analyses were mainly focused on the differences between blind-side hypermelanotic skin tissues (BH) and blind-side normal skin tissues(BN).

In total, 160 DEGs were identified in BN-vs-BH comparative group,then these DEGs were further analyzed by GO terms and KEGG pathway enrichments to assist in understanding potential molecular mechanism of hypermelanosis.In GO analyses, melanin metabolic process, DNA binding and cytoplasmic vesicle membrane was the predominant categories.In KEGG pathway analyses, tyrosine metabolism was the most significantly enriched category.According to these results,tyrandtyrp1as tyrosine metabolism-related genes are regard as key genes which play an important role in pigment metabolic process.In human, oculocutaneous albinism (OCA) is commonly subdivided into four types based on the genes that are mutated: TYR, OCA2, TYRP1, and SLC45A2 (Tomita,Takeda, Okinaga, Tagami, & Shibahara, 1989; Rinchik et al., 1993;Boissy et al., 1996; Newton et al., 2001).In the present study, these four genes were all significantly up-regulated in both ocular-side normal skin and blind-side hypermelanotic skin, while their expression in blind-side normal skin was very low (Supplementary Fig 2; Supplementary Table 5).Albinism in fish as well as other mammals (including humans),in most cases, are caused by mutations of key genes of the melanogenesis pathway, such as occurs in medaka (Koga, Inagaki, Bessho, & Hori,1995), zebrafish (Haffter et al., 1996), rainbow trout (Boonanuntanasarn, Yoshizaki, Iwai, & Takeuchi, 2004), cavefish (Protas et al., 2006)and channel catfish (Li et al., 2017).Therefore, from this perspective,the molecular mechanisms of albinism and hypermelanosis may be very different.

According to previous studies and our knowledge, blind-side hypermelanosis in flatfish should be ascribed to environmental factor(s).Specifically, there must be some factor playing a stimulating role so that key genes of the melanogenesis pathway are activated.However,the trigger is still unclear.Recently, studies revealed that melanosomal transmembrane proteins such as OCA2 and SLC45A2 modulate melanosomal pH and contribute to the activity of tyrosinase (TYR), an essential protein for melanin production (Bellono & Oancea, 2014).Mutations and polymorphisms of some protein molecules (e.g., OCA2 and SLC45A2) result in variations of skin and eye color as well as ocular and dermatological diseases such as retinal pigment epitheliopathy,albinism and hyperpigmentation disorders in human (Chi et al., 2006;Fistarol & Itin, 2010; Pavan & Sturm, 2019).In the present study,oca2andslc45a2were significantly up-regulated in blind-side hypermelanotic skin while in blind-side normal skin the relative expression levels were very low.These retrospective and inferential results may suggest that abnormal pH values and/or ion concentrations of seawater result in disorders in melanosomes, where membrane transport proteins(such as OCA2 and SLC45A2) may serve as a “switch” for melanogenesis in tongue sole.However, studies ofoca2andslc45a2are still very limited in fish and their relationship with tyr are still unclear.There is evidence that the mutation ofoca2orslc45a2resulted in albinism in zebrafish (Danio rerio) (Beirl, Linbo, Cobb, & Cooper, 2014), cavefish(Astyanax mexicanus) (Klaassen, Wang, Adamski, Rohnerb, & Kowalko,2018) and medaka (Oryzias melastigma) (Jeong et al., 2020).Further studies are needed to elucidate the molecular mechanisms of hypermelanosis in tongue sole.Our results may imply that blind-side hypermelanosis is an environmental regulation of gene expression and adaptation in flatfish.

5.Conclusion

In conclusion, based on comparative transcriptome analyses of hypermelanotic and normal skin tissues, many hypermelanosis-related genes were mined, which contribute to the understanding of the molecular mechanisms of hypermelanosis in tongue sole.Membrane transport proteins (such as OCA2 and SLC45A2) may serve as a “switch”for melanogenesis.Our results provided valuable breeding and genetic resources for molecular studies of pigmentation formation in tongue sole.

CRediT authorship contribution statement

Yangzhen Li: Methodology, Conceptualization, Formal analysis,Funding acquisition, Writing - original draft, Supervision.Peng Cheng:Resources, Testing and assaying, Investigation, Writing - original draft.Ming Li: Testing and assaying, Data curation, Writing - review & editing.Yuanri Hu: Resources, Investigation, Validation.Zhongkai Cui:Resources, Investigation.Chuantao Zhang: Resources, Investigation.Songlin Chen: Funding acquisition, Resources, Writing - review &editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31702333), China Agriculture Research System (CARS-47-G03),Central Public-interest Scientific Institution Basal Research Fund, CAFS(2020TD20), AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology(No.2017ASTCP-OS15) and Taishan Scholar Climbing Project of Shandong.

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aaf.2021.02.003.