As one of the most diverse phenotypic characteristics in vertebrates, coloration plays numerous adaptive functions like camouflage, predator deterrence and species recognition [1
]. Skin coloration can be influenced by many factors, such as genetics, diet, environmental or healthy condition, etc.
]. Nevertheless, genetic is still the major determination, the kind of pigmentation related genes and their variant expressions are the major reason of diverse form coloration [1
]. In mammalian systems, melanophores are the only chromatophore type found in their skin. In contrast, several kinds of chromatophores are found take part in the formation of variety coloration in teleost, including melanophores (melanin granules), xanthophores (pteridine or carotenoid granules), iridophores (guanine), leucophore (unknown) and erythrophores (carotenoids and pteridine) [4
]. As most of the pigment related genes were first identified in laboratory mice (genus Mus.
), to date, most of the known pigmentation genes are genes responsible for producing melanin [10
], even in the teleosts system. Only a few studies about genetic of xanthophore [16
] and iridophore [17
] have been reported recently. However, pigmentation is an important economic trait for fish, achieving a uniform and bright coloration is crucial for fish farms.
With the advantage of low cost and speed, massively parallel sequencing (Illumina) RNA-Seq analysis is now the most convenient method to find out new genes and investigate gene expression patterns of non-model organisms, especially for species of which the whole genome sequence is not yet available, such as sheep [19
], spider [20
], Ischnura elegans
], Yesso Scallop [23
To date, several studies have reported on the gene expression profile of different coloration patterns of fresh-water fish like common carp [3
], cichlids [25
] and zebrafish [17
]. These studies have found that signaling pathway such as Wnt (wingless-type MMTV in integration site family), MAPK (mitogen-activated protein kinase) and cAMP (cyclic adenosine monophosphate) were conserved melanin-synthesis related pathways in vertebrates. Higdon et al.
] have proposed the purine synthesis and phosphoribosyl pyrophosphate might take part in the guanine production in zebrafish, the latter is a basic component of iridophore. However, studies about the genetic profiles on the skin of seawater fish species remain scarce. Considering from the morphology perspective, body coloration differences were mainly caused by the type, density and distribution of chromatophores [5
]. From the cellular level, which chromatophores and how they involved in the formation of variety colorations, and from the genetic level, which genes correlated with the different pigmentations is still poorly understood.
In the South China Sea (SCS), there are about 20 indigenous species of genus Lutjanus
present, which are economically important and a significant source of food for developing countries around SCS [26
]. All of them have diagnostic color patterns that are primary taxonomic identification characters. To date, most studies about Lutjanus
were mainly focused on their phylogenetic relationships [28
]. Interestingly, Wang et al.
] have found that as a kind of coral reef fish, there might be some relevance between the coloration and speciation in Lutjanus
. However, there is little knowledge about the formation of these diverse pigment patterns in Lutjanus
. Crimson snapper (Lutjanus erythropterus
), which is distributed over the Indo-West Pacific and habitats throughout coral reef and hard-bottom, is one of the most economically important fish of SCS [30
]. A suitable model for exploring the genetic basis of skin coloration is provided by the distinct skin colors of crimson snapper. The morphological characters of crimson snapper are very conservative and simple—the whole body is light red with more intense pigment on the back and a big black dot on the caudal trunk. To better understand the cells and genetic factors that influence the pigmentation formation, in this study, we utilized Stereomicroscope and Transmission Electron Microscopy (TEM) technology to observe the chromatophores morphology of black skin and red skin in crimson snapper. RNA-Seq was conducted on the two color skins of crimson snapper to compare their gene expression profiles. The purpose of this study is to provide basic information about the color difference from the cellular level, and identify the genes potentially related to the color determination of crimson snapper as well as find out the genetic differences between the two different color traits. Understanding this will not only enrich the information of skin color varieties in fish but also help the selection of fish species with consumer-favored coloration from the genetic level. On the other hand, our ultimate aim is to provide some candidate pigmentation genes to investigate the correlation between coloration and sympatric speciation in Lutjanus
Animal coloration plays important roles in communication, ecological interactions and even speciation [16
]. Studies have found that diverse body colorations are mainly controlled by the development and patterning of pigment cells, of which genetic was the major determination. Thus, in this study, firstly, we observed the morphology of black skin and red skin of crimson snapper. Secondly, by Illumina sequencing technology, we studied the genetic profiles of black skin and red skin of crimson snapper from transcriptome level, and by contrast, 9200 significantly DTGs were found. The identified DTGs between the two color skins will not only help us to understand the molecular mechanism of skin color differences, but also provide us valuable gene information for exploring the relevance of speciation and pigmentation in this species. Compared to the recent RNA-Seq research on common carp skin [33
], more DTGs were found in our study. The first reason might be less use of analysis technology, as the aim of this study is to identify a large number of candidate genes for subsequent analysis, only one conventional technology DEGseq [34
] was used to identify the DTGs. The second reason might be the diverse chromatophores composition of crimson snapper skins, as shown in Figure 1
In this study, we found ribosome protein genes were accounted for the highly expressed genes in each tissue, which suggested ribosome proteins might play an important role in the fish skin formation. Previous study has reported that in the transcriptome of zebrafish pigment cells, four of the top five most highly expressed genes were ribosomal proteins [17
]. A similar finding was also reported on the transcriptome analysis of sheepskin [19
]. Studies have proved that highly expressed levels of ribosome proteins related genes not only revealed the high rates of protein translation in organism [35
], but also have some correlation with the mouse black coat color [36
]. Combined with the more highly expressed of ribosome protein genes in black skin, so we inferred the ribosomal proteins genes might involve in the formation of the black skin coloration of crimson snapper. However, further studies are needed to deliberate its exact function. While, different from the black skin, in the red skin, creatine kinase M-type, fructose-bisphosphate aldolase (FBA
), cytochrome c oxidase subunit 1 (COX1
), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH
), NADH dehydrogenase subunit 5 (NADH5
) and parvalbumin were also showed very high expression, all of these genes were found to be iridophore-related genes in zebrafish [17
]. Meanwhile, Fan et al.
] has found that the expression of NADH5
were very high in the white skin of sheep, as both of them were genes encoding for the enzymes responsible for oxidative and dehydrolytic, so they presumed that the high expression of them might imply strong metabolism characteristic of iridophores. However, the function of the NADH5
in crimson snapper needs further elucidation. As an activator to tyrosine kinase [37
], fructose-bisphosphate aldolase (FBA
) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH
) were constituents of the glycolytic/gluconeogenesis pathway. However, from the recent study [17
], we known that GAPDH
might play important roles from the guanine synthesis to the formation of iridophore pigment contained organelles. All of these results indicated these genes might play some role in the red skin in the energy metabolism or synthesis of guanine, further work is still needed to determine how these genes function.
After the GO and KEGG analyses of DTGs, the most clustered groups of DTGs were consistent with previous works about fish, like zebrafish [17
], Midas cichlids [25
] and common carp [3
]. After the GO analysis of the DTGs, we found that most of the down-regulated genes were enriched in pathways like Glycolysis/Gluconeogenesis, Citrate cycle, Oxidative phosphorylation, Cardiac muscle contraction and Proteasome, which implied the participation of these pathways in the formation of red skin. In zebrafish, several TEM studies about fish skin have showed the existences of stacks of guanine plates in iridophores [5
], and glycolysis and citrate cycle pathway were found to be key participators with the extensive guanine synthesis [17
]. Combined with our TEM results (Figure 1
d), a number of reflecting plates were found in the iridophores; thus, higher expression of genes with these pathways might be in accordance with the higher requirement of guanine for the reflective iridophore pigment in red fish skin crimson snapper. Further work is still needed to determine how these pathways coordinate the up-regulation of guanine synthesis in iridophores.
To test the reliability of the RNA-Seq datas, 14 genes were chosen randomly for qRT-PCR, including WNT5a
, the tyrosine gene family (DCT
As shown in Figure 4
, the expression pattern of the pigment-specific genes from qRT-PCR was almost in accordance with the RNA-Seq result except the MITFb
gene, and the expression level of the two methods was nearly in accordance, with only slight differences, indicting the reliability of the transcriptome data. Several studies [24
] have reported that WNT signaling pathway taken part in the synthesis of melanogenesis in teleost as well as mammals. WNT5a
, a non-canonical Wnt protein family gene which was found located in the matrix and precortex cells in the hair follicles of mice [42
], was 1.1-fold up-regulated in the black skin. Interestingly, in the recently RNA-Seq study about the common carp skin, the member of Wnt protein family showed higher expression in YRC was WNT5b
. Brassch et al.
] have found that due to the fish-specific genome duplication (FSGD), 75% of the melanogenic enzymes were found to be duplicated, and three different fated for duplicated genes were observed in [45
]. So further studies should be conducted to determine whether species-specific evolution variation exist in WNT5
duplicates in crimson snapper. MITF
], the master regulator of melanophore identity, was 1.2-fold up-regulated in the black skin, and the downstream gene of MITF
, tyrosinase gene family [44
], which is well known to take part in the enzymatic conversion of tyrosine to melanin, were also showed up-regulation in the black skin. However, agouti signaling protein (ASIP
], the gene blocked the melanogenesis by antagonizing the binding of α-MSH
, showed a higher expression in red skin in either method. All of these results not only proved the credibility of the transcriptome data but also indicted the conservation of these pigmentation genes in teleost.
According to the comparison between known zebrafish pigmentation genes with the transcriptome data of this study, majority of the pigmentation genes could be found in the snapper fish. In addition, we found most of the known pigmentation genes have shown significantly different expression patterns between the two tissues. Such as sodium calcium transporter 45a2 (SLC45a2
) showed higher expression in black skin in contrast with red skin, with OCA2
, sodium calcium transporter 24a5 (SLC24a5
), pre-melanosomal protein a (PMELa
) and sox9b were followed. Studies have found SLC45a2
performed some role for organelle pH homestsis in pigment cells [49
] and in situ
analyses have also revealed its enriched expression in melanophores in zebrafish [49
, together with the SLC45a2
also belongs to the typical melanin synthesis pathway [52
, encodes for a pigment cell-specific protein that might take part in the formation of fibrillar sheets contained in the melanosome [53
]. In former RNA-Seq studies on cichlids [25
] and stickleback [54
], the expression of these genes was also found to be higher in dark bars. These results revealed the conservation of pigmentation genes across various species in term of sequences and functions.
From the cellular level, we found that the differences in the two colorations depending primarily upon the density and distribution of the chromatophores, in the black skin melanophores account for the major, and in the red skin leaving the iridophores and xanthophores the major. However, from the genetic level, after analyzing of the two skin transcriptomes, different expressed candidate pigmentation genes were mainly enriched in the pathways of melanin and guanine synthesis. There might be two reasons to explain this: Firstly, in crimson snapper, the xanthophores mainly filled with carotenoid droplets, which is a kind of pigment that vertebrates cannot synthesis endogenously [16
]. Secondly, like Ng et al.
] has found in Nothobranchius
fish, xanthophores were functionally more related to the melanophores and most likely ontogentically closer to the melanocyte lineage than the iridophores.
In this study, approximately 50% of the DTGs did not find significant matches with known proteins after BLAST in public databases. The similar result has also occurred in the common carp skin transcriptome [3
]. One of the possible elucidations might be that the non-model species possess many potential novel genes or transcripts that cannot be found in public databases. Therefore, further characterization of the unknown DTGs identified in the present study is required.