Pigment Identification and Gene Expression Analysis during Erythrophore Development in Spotted Scat (Scatophagus argus) Larvae

Abstract

Red coloration is considered an economically important trait in some fish species, including spotted scat, a marine aquaculture fish. Erythrophores are gradually covered by melanophores from the embryonic stage. Despite studies of black spot formation and melanophore coloration in the species, little is known about erythrophore development, which is responsible for red coloration. 1-phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to inhibit melanogenesis and contribute to the visualization of embryonic development. In this study, spotted scat embryos were treated with 0.003% PTU from 0 to 72 h post fertilization (hpf) to inhibit melanin. Erythrophores were clearly observed during the embryonic stage from 14 to 72 hpf, showing an initial increase (14 to 36 hpf), followed by a gradual decrease (36 to 72 hpf). The number and size of erythrophores at 36 hpf were larger than those at 24 and 72 hpf. At 36 hpf, LC-MS and absorbance spectrophotometry revealed that the carotenoid content was eight times higher than the pteridine content, and β-carotene and lutein were the main pigments related to red coloration in spotted scat larvae. Compared with their expression in the normal hatching group, rlbp1b, rbp1.1, and rpe65a related to retinol metabolism and soat2 and apoa1 related to steroid hormone biosynthesis and steroid biosynthesis were significantly up-regulated in the PTU group, and rh2 associated with phototransduction was significantly down-regulated. By qRT-PCR, the expression levels of genes involved in carotenoid metabolism (scarb1, plin6, plin2, apoda, bco1, and rep65a), pteridine synthesis (gch2), and chromatophore differentiation (slc2a15b and csf1ra) were significantly higher at 36 hpf than at 24 hpf and 72 hpf, except for bco1. These gene expression profiles were consistent with the developmental changes of erythrophores. These findings provide insights into pigment cell differentiation and gene function in the regulation of red coloration and contribute to selective breeding programs for ornamental aquatic animals.

Keywords: erythrophore; gene expression; pigment; spotted scat.

Figure 1

Microscopic observation of erythrophores in spotted scat during hatching. Control, control group; Treatment, PTU-treated group. (a,c,e,g,i,k,m,o) are the control group from 14–72 h after fertilization, and (b,d,f,h,j,l,n,p) are the PTU-treated group from 14–72 h after fertilization. Erythrophores are indicated by white arrows (scale bar, 200 μm).

Figure 2

Characterization of DEGs between the control and PTU treatment groups. (A) PCA shows the differences between groups. (B) In the volcano plot, green dots represent down-regulated genes, red dots represent up-regulated genes, and black dots indicate genes without a significant difference. (C,D) GO enrichment analysis of DEGs, C, GO terms for up-regulated genes; D, GO terms for down-regulated genes. The circles represent the gene ratio, and the colors represent the p value, respectively.

Figure 3

KEGG pathway enrichment analysis of DEGs between the control and PTU treatment groups. The size of the black circle represents the number of DEGs. The colors represent the q-value. The triangle, inverted triangle, and circle represent up-, down-, and up- and down-regulated genes, respectively.

Figure 4

Gene expression validation by RNA-Seq (A) and qRT-PCR (B). (A) FPKM values, (B) Relative mRNA expression levels. The green and blue columns represent the control and PTU treatment groups, respectively. The relative expression levels of mRNA transcripts were detected using qRT-PCR via the 2^−∆∆Ct method. Data are expressed as means ± standard error (SE) (n = 3). β-actin was used as the reference gene. The symbols “**” above the error bars indicate significant differences at the levels of p = 0.01.

Figure 5

Characteristics (A–C) and pigment types and contents (D,E) responsible for red coloration in spotted scat larvae. (A) Observation of carotenoid droplets of erythrophores at 24, 36, and 72 h after fertilization. (b,e,h) are the high magnification of erythrophores in the head (white boxed area in (a,d,g)), and (c,f,i) are the high magnification of tail part (red boxed area in (a,d,g)). Erythrophores are indicated by white arrows. (B) Numbers of erythrophores at different stages (black box in (a,d,g) of panel (A)). (C) Diameters of erythrophores at different stages. Red, green, and blue columns represent the data at 24, 36, and 72 h, respectively. (D) Identification of pigment types responsible for the red coloration by LC–MS. “Standard” refers to the pigment standards, and “Tested” represents the spotted scat larvae sample. (E) Pteridine and carotenoid concentrations at 36 hpf were determined using spectrophotometry (n = 3). The red and yellow columns represent the concentration of carotenoids and pteridines, respectively. Data are presented as means ± SE. The symbols “*” and “**” above the error bars indicate significant differences at the levels of p = 0.05 and p = 0.01, respectively.

Figure 6

Expression of genes responsible for red coloration in spotted scat larvae at different stages. Red, green, and blue columns represent gene expression at 24, 36, and 72 h, respectively. Relative expression levels were detected using qRT-PCR via the 2^−∆∆Ct method. Data are expressed as means ± SE (n = 3). β-actin was used as the reference gene. The symbols “*”, “**” and “ns” above the error bars indicate significant differences at the levels of p = 0.05, p = 0.01 and no significant difference, respectively.

Figure 7

Predictive model for the regulation of erythrophore development and carotenoid metabolism in spotted scat larvae. Genes related to carotenoid metabolism are highlighted by orange circles, and the genes involved in pteridine synthesis are marked by yellow circles.