Establishment and characterization of a golden pompano (Trachinotus blochii) fin cell line for applications in marine fish pathogen immunology.

Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20% fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20% FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1, irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88, irak1, nfκb, il1ß, il6, and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology.

Molecular and functional characterization of golden pompano (Trachinotus blochii) TBK1 on IFN regulation.

The golden pompano (Trachinotus blochii), a pivotal commercial marine species in China, has gained significant popularity worldwide. However, accompanied with rapid growth and high density aquaculture, golden pompano has been seriously threatened by Nervous necrosis virus (NNV), while its molecular biology research regarding the innate immune system remains unexplored, which is crucial for understanding the activation of interferon (IFN) production and antiviral responses. In this study, we aimed to identify the characterization and function of golden pompano TANK-binding kinase 1 (gpTBK1), thereby providing evidence of the conservation of this classical factor in the RLR pathway among marine fish. Initially, we found the expression of gpTBK1 upregulation in diseased golden pompano with NNV infection and we successfully cloned the full-length open reading frame (ORF) of gpTBK1, consisting of 2172 nucleotides encoding 723 amino acids, from the head kidney. Subsequent analysis of the amino acid sequence revealed homology between gpTBK1 and other fish TBK1 proteins, with conserved N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD). Moreover, the expression pattern showed that gpTBK1 exhibited ubiquitous expression across all evaluated tissues. Furthermore, functional identification experiments indicated that gpTBK1 activated interferon promoters' activity in golden pompano and induced the expression of downstream IFN-stimulated genes (ISGs). Notably, gpTBK1 was found to co-localize and interact with gpIRF3 in the cytoplasm. Collectively, these data provide a comprehensive analysis of the characterization and functional role of gpTBK1 in promoting interferon production. This research may facilitate the further study of the innate antiviral response, particularly the anti-NNV mechanisms, in golden pompano.

Transcriptome analysis reveals candidate miRNAs involved in skin color differentiation of juvenile Plectropomus leopardus in response to different background colors.

Red skin color in Plectropomus leopardus is important to its ornamental and economic value. However, the color of P. leopardus can change during the rearing process, darkening and turning black due to the influence of environmental background color. The underlying molecular mechanisms that regulate this phenomenon remain unclear. MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play important roles in numerous biological processes, such as skin differentiation and color formation in many animals. Therefore, we performed miRNA sequencing of P. leopardus skin before (initial) and after rearing with three different background colors (white, black, and blue) using Illumina sequencing to identify candidate miRNAs that may contribute to skin color differentiation. In total, 154,271,376 clean reads were obtained, with over 92 % of them successfully mapped to the P. leopardus reference genome. The miRNA length distributions of all samples displayed peaks around a typical length of 22 nt. Within these sequences, 243 known and 287 novel miRNAs were identified. A total of 65 significantly differentially expressed miRNAs (DEMs) were identified (P < 0.05), including 40 known DEMs and 25 novel DEMs. These DEMs included novel_561, miR-141-3p, and miR-129-5p, whose target genes were primarily associated with pigmentation related processes, including tyrosine metabolism, melanogenesis, and the Wnt signaling pathway. These findings shed light on the potential roles of miRNAs in the darkening of skin color in P. leopardus, thus enhancing our understanding of the molecular mechanisms involved in skin pigmentation differentiation in this species.

The muscle nutritional components analysis of golden pompano (Trachinotus blochii) in different mariculture area, growth stages, and genders.

Golden pompano (Trachinotus blochii) is an economically important fish which exhibits sexual size dimorphism and is widely cultivated in the southern seas of China. To evaluate the nutritional composition of T. blochii of different mariculture areas, growth stages, and genders, the moisture, ash, amino acids, and fatty acids in the muscle were measured using national standard biochemical assay. The analysis found 16 kinds of amino acids in the muscle of T. blochii. The EAA contents of fish from Guangdong (GD) and Guangxi (GX) were significantly lower than those of Hainan (HN) and Fujian (FJ) (p < 0.05). The unsaturated fatty acids were higher in T. blochii cultured in HN and FJ (p < 0.05). Within the same sea area, the contents of TAA, EAA, DAA, and PUFA increased with growth in T. blochii, but the differences were not significant (p > 0.05). EAA/TAA and EAA/NEAA conformed to the ideal FAO/WHO model. The AAS, CS, and EAAI scores of amino acids within groups gradually increased with growth. The TAA, EAA and PUFA contents in females were higher than in males (p > 0.05). The slightly higher amounts of amino acids and fatty acids in female T. blochii indicated females had higher nutritional value. In conclusion, the HN and FJ groups, the later growth stages, and the female T. blochii had generally higher nutritional values than their respective counterparts. These results provide fundamental data supporting all-female T. blochii breeding and culture, and optimized marketing body size.

Weighted correlation network analysis of the genes in the eyes of juvenile Plectropomus leopardus provide novel insights into the molecular mechanisms of the adaptation to the background color.

Plectropomus leopardus is a valuable marine fish whose skin color is strongly affected by the background color. However, the influence of the visual sense on the skin color variation of P. leopardus remains unknown. In the present study, transcriptome analysis was used to examine the visual response mechanism under different background colors. Paraffin sections of the eyes showed that the background color caused morphological changes in the pigment cells (PCs) and outer nuclear layer (ONL) and the darkening of the iris color. The transcriptome analysis results indicated that the gene expressions in the eyes of P. leopardus were significantly different for different background colors. We identified 4845, 3069, 5874, and 6309 differentially expressed genes (DEGs) in the pairwise comparisons of white vs. initial, blue vs. initial, red vs. initial, and black vs. initial groups, respectively. Some hub genes and key pathways regulating the adaptive mechanism of P. leopardus's eyes to the background color were identified, i.e., the JAK-STAT, mTOR, and Ras signaling pathways, and the ndufb7, slc6a13, and novel.3553 gene. This adaptation was achieved through the synthesis of stress proteins and energy balance supply mediated by hub genes and key pathways. In addition, the phenylalanine metabolism, tyrosine metabolism, and actin cytoskeleton-related processes or pathways and genes were responsible for iris and skin color adaptation. In summary, we inferred that stress protein synthesis, phenylalanine metabolism, and energy homeostasis were critical stress pathways for P. leopardus to adapt its skin color to the environment. These new findings indicate that the P. leopardus skin color variation may have been caused by the environmental adaption of the eyes. The results provide new insights into the molecular mechanisms underlying the skin color adaptation of P. leopardus.

Transcriptome analysis of differentially expressed circular RNAs in the testis and ovary of golden pompano (Trachinotus blochii).

The artificial breeding of golden pompano (Trachinotus blochii) has expanded greatly in recent years, and after long-term breeding efforts, clear sexual dimorphisms have been observed in T. blochii growth traits, with females growing faster. As sponges of microRNA (miRNAs), circular RNAs (CircRNAs) can alleviate miRNA inhibition of target mRNA. However, few studies have examined sex-related CircRNAs and none of those have looked at T. blochii. To further understand the role of CircRNAs in sex differentiation and sexual size dimorphism in T. blochii, six CircRNA libraries were constructed from the testes and ovaries of T. blochii. A total of 1522 CircRNAs were found distributed over all 24 chromosomes of T. blochii. 135 differentially expressed CircRNAs (DECs) were identified by screening, These DECs were then subjected to GO enrichment, which found 47 enriched pathways. A number of CircRNAs were enriched in cellular processes and metabolic processes. According to the KEGG pathway analysis, a series of sex differentiation pathways were enriched, including the GnRH, calcium, and MAPK signaling pathways. Furthermore, we selected two CircRNAs from the DECs named circ-cacna1b and circ-octc. We found that the cacna1b gene is regulated by 7 miRNAs, 3 of which were regulated by circ-cacna1b, i.e., mmu-miR-138-5p, fru-miR-138, and pma-miR-138b. In addition, the miRNA named pma-miR-138b can regulate sex-related genes, such as sox9 and dmrt1, among others. The co-expression network of CircRNA-miRNA-mRNA showed circ-cacna1b may play a crucial role in T. blochii sex differentiation by regulating pma-miR-138b to affect the expression of sex differentiation genes. The circ-octc may be one of the largest contributors to sexual size dimorphism during growth through its effect on lipid metabolism. These findings could broaden our understanding of CircRNAs and provide new insight into their function in sex differentiation and growth.

Identification and Characterization of Sex-Biased miRNAs in the Golden Pompano (Trachinotus blochii).

The golden pompano (Trachinotus blochii) is a marine fish of considerable commercial importance in China. It shows notable sexual size dimorphism; the growth rate of females is faster than that of males. Therefore, sex-biased research is of great importance in T. blochii breeding. However, there have been few studies on sex differentiation and mechanisms underlying sex determination in T. blochii. MicroRNAs (miRNAs) play crucial roles in sex differentiation and determination in animals. However, limited miRNA data are available on fish. In this study, two small RNA libraries prepared from the gonads of T. blochii were constructed and sequenced. The RNA-seq analysis yielded 1366 known and 69 novel miRNAs with 289 significantly differentially expressed miRNAs (p < 0.05). Gene ontology (GO) analysis confirmed that the TFIIA transcription factor complex (GO: 0005672) was the most significantly enriched GO term. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the differentially expressed miRNAs and target genes were mainly related to sex determination and gonadal developmental signaling pathways, specifically the Wnt signaling pathway, MAPK signaling pathway, and steroid biosynthetic pathway. MiRNA-mRNA co-expression network analysis strongly suggested a role for sex-biased miRNAs in sex determination/differentiation and gonadal development. For example, gata4, foxo3, wt1, and sf1 genes were found to be regulated by bta-miR-2898; esr2 and foxo3 by novel_176, and ar by oar-let-7b. Quantitative real-time polymerase chain reaction analysis of selected mRNAs and miRNAs validated the integrated analysis. This study established a set of sex-biased miRNAs that are potential regulatory factors in gonadal development in T. blochii. These results provide new insight into the function of miRNAs in sex differentiation and determination in T. blochii and highlight some key miRNAs for future studies.

The Effect of Background Color on Skin Color Variation of Juvenile Plectropomus leopardus.

Fish skin color is usually strongly affected by the background color of their environment. The study investigated the effects of five different background colors on the skin color of leopard coral groupers (Plectropomus leopardus). More than 450 juveniles were reared in Blue, Red, Black, White, and Transparent background tanks for 56 days. The paraffin section showed that the skin melanin zone of fish in the White group was smaller, whereas the Black and Red groups (especially Black) were nearly the largest. The apparent skin color of P. leopardus was red on the white background, which darkened in response to the other color backgrounds. The Black group revealed the blackest skin color, followed by the transparent group. Moreover, the White group had the highest L*, a*, and b* values. The melanin content and tyrosinase activity in the dorsal and ventral skin of the Black group were significantly higher than those in the other groups (p < 0.05), and the serum α-MSH level was higher in the Black group as well. The carotenoid and lutein contents showed completely different trends among the experimental groups, as carotenoid content was higher in the Red and White groups, while lutein content was higher in the Transparent group. The expression level of scarb1 was highest in the Blue and White groups, followed by the Transparent group, and lowest in the Black group (p < 0.05). The expression trend of scarb1 was similar to the skin color in different backgrounds, indicating that the background color regulated scarb1 expression level through visual center, then influenced the uptake and transport of carotenoids, then influenced the skin color formation of P. leopardus. Moreover, lighter colors inhibited the formation of melanocytes and had a significant effect on carotenoid and lutein contents. Pigment-related genes were involved in the regulation of fish skin color, and they were affected by background color in P. leopardus. These results indicate that a white background is more conducive to maintaining red skin color in juvenile P. leopardus.

Transcriptome Analysis Reveals the Complex Regulatory Pathway of Background Color in Juvenile Plectropomus leopardus Skin Color Variation.

Fish skin color is often strongly affected by background color. We hypothesized that the regulatory mechanism of variations in skin color in P. leopardus is linked to the background color. In this study, we conducted transcriptome analysis of Plectropomus leopardus cultured under different background colors to compare gene expression levels and the important signaling pathways. The RNA-seq analysis yielded 26,675 known mRNAs, 3278 novel mRNAs, and 3179 differentially expressed genes (DEGs). The DEGs related to melanin synthesis were screened out. Some key melanin-related genes were identified, specifically tyr, slc7a11, mc1r, ednrb, dct, tat, and wnt1. These DEGs were mainly involved in melanogenesis, including tyrosine metabolism, the Wnt signaling pathway, and the cAMP signaling pathway. The expression levels of some key genes were upregulated when background color deepened, such as α-msh, wnt, and gf. The α-MSH/cAMP-dependent, Wnt/ß-catenin, and PI3K/Akt signaling pathways were activated, resulting in the accumulation of intracellular mitf. mitf promoted melanin production by binding to the tyr/tyrp1/dct promoter region. In the present study, we explored the molecular mechanism underlying the darkened skin color pattern of P. leopardus, providing a theoretical basis for the molecular mechanism underlying pigmentation in P. leopardus.

Identification of SF-1 and FOXL2 and Their Effect on Activating P450 Aromatase Transcription via Specific Binding to the Promoter Motifs in Sex Reversing Cheilinus undulatus.

The giant wrasse Cheilinus undulatus is a protogynous socially hermaphroditic fish. However, the physiological basis of its sex reversal remains largely unknown. cyp19 is a key gender-related gene encoding P450 aromatase, which converts androgens to estrogens. cyp19 transcription regulation is currently unknown in socially sexually reversible fish. We identified NR5A1 by encoding SF-1, and FOXL2 from giant wrasse cDNA and cyp19a1a and cyp19a1b promoter regions were cloned from genomic DNA to determine the function of both genes in cyp19a1 regulation. Structural analysis showed that SF-1 contained a conserved DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD). FOXL2 was comprised of an evolutionarily conserved Forkhead domain. In vitro transfection assays showed that SF-1 could upregulate cyp19a1 promoter activities, but FOXL2 could only enhance cyp19a1b promoter transcriptional activity in the HEK293T cell line. Furthermore, HEK293T and COS-7 cell lines showed that co-transfecting the two transcription factors significantly increased cyp19a1 promoter activity. The -120 to -112 bp (5'-CAAGGGCAC-3') and -890 to -872 bp (5'-AGAGGAGAACAAGGGGAG-3') regions of the cyp19a1a promoter were the core regulatory elements for SF-1 and FOXL2, respectively, to regulate cyp19a1b promoter transcriptional activity. Collectively, these results suggest that both FOXL2 and SF-1 are involved in giant wrasse sex reversal.

Characterization and functional analysis of myostatin and myogenin genes involved in temperature variation and starvation stress in Golden pompano, Trachinotus blochii.

Animal growth and development is a complicated process and is regulated by multi-genes. Myostatin (Mstn) and myogenin (Myog) are a pair of negative and positive regulators respectively, which play an important role in the generation of muscle cells. In order to study the function of these two genes in muscle growth of Trachinotus blochii, full lengths of two mstn genes (mstn-1 and mstn-2) and myog gene were cloned using RACE. We first identified and characterized the complete cDNA sequences of mstn-1, mstn-2, and myog genes derived from T. blochii, an economically important mariculture species in China. Multiple sequence alignment of amino acids and phylogenetic analysis revealed that the Mstn and Myog were highly conserved to the other Perciformes. In addition, gene duplication of mstn in T. blochii was observed. mstn-1 mRNA was mainly expressed in the muscle and gonad, while mstn-2 and myog transcripts were detectable mainly in the brain and muscle, respectively. Moreover, the nutritional status and temperature influenced abundance levels in brain and muscle. Results suggested that mstn and myog genes play an important role in muscle growth of T. blochii, mstn may not be limited to control of muscle growth in fish and could also be involved in other biological functions.

Identification of Candidate Genes Associated With Hypoxia Tolerance in Trachinotus blochii Using Bulked Segregant Analysis and RNA-Seq.

Golden Pompano (Trachinotus blochii) has rapidly developed into the one of the main valuable fish species in Chinese marine aquaculture. Due to its rapid growth, active metabolism, and high oxygen consumption, hypoxia will increase its mortality and cause serious economic losses. We constructed two experimental groups of fish with different degrees of tolerance to hypoxia, used BSR-Seq analysis based on genome and genetic linkage groups to locate SNPs and genes that were related to the differences in hypoxia tolerance. The results showed that hypoxia tolerance SNPs of golden pompano may be jointly determined by multiple linkage groups, especially linkage groups 18 and 22. There were 768 and 348 candidate genes located in the candidate regions of the brain and liver, respectively. These genes were mainly involved in anaerobic energy metabolism, stress response, immune response, waste discharge, and cell death. The prostaglandin-endoperoxide synthase 2 (PTGS2) on LG8, which is involved in the metabolism of arachidonic acid, has a G/A nonsynonymous mutation at position 20641628, and the encoded amino acid was changed from hydrophobic aspartic acid to asparaginate. The specific pathway of the RIG-I-like receptor signaling pathway in the liver may mediate the metabolic system and the immune system, linking glucose metabolism with immune regulation. The death of the hypoxia-intolerant group may be due to the accumulation of lactic acid caused by the activation of anaerobic glycolysis during the early stage of hypoxia stress, and the activation of type I interferon was inhibited, which resulted in decreased immunity. Among the genes involved in the RIG-I-like receptor signaling pathway, the CYLD Lysine 63 Deubiquitinase (CYLD) located on LG16 had a G/T nonsynonymous mutation at position 13629651, and the encoded amino acid was changed from alanine acid to valine. The interferon induced with helicase C domain 1 (Ifih1) located on LG18 has a G/C nonsynonymous mutation at position 16153700, and the encoded hydrophilic glycine was changed to hydrophobic alanine. Our findings suggest these SNPs may assist in the molecular breeding of hypoxia-tolerant golden pompano, and speculate that the balance of glucose and lipid metabolism plays a key role in Trachinotus blochii under acute hypoxia.

The stage-specific long non-coding RNAs and mRNAs identification and analysis during early development of common carp, Cyprinus carpio.

Cyprinus carpio is considered an alternative vertebrate fish model to zebrafish. However, systemic times-series research on the lncRNAs and mRNAs during early development of C. carpio has not been reported yet. This study provides the first long non-coding RNA (lncRNA)-mRNA expression profiles during six main early development stages (2 h post-fertilization hpf, 6 hpf, 12 hpf, 20 hpf, 64 hpf and 1 day post-hatching). A total of 51,979 lncRNAs were identified. We screened the top 10 abundance lncRNAs and mRNAs and stage-specific lncRNAs and mRNAs (specificity measure SPM > 0.9). We identified significant differentially expressed lncRNAs and mRNAs (|log2 (fold change)| ≥ 1 and false discovery rate FDR of <0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous signaling pathways. Additionally, the lncRNA-mRNA co-regulated network analysis of two lncRNAs (lncrps25 and malat1) and two mRNAs (mitf and troponin T) were investigated. Our results provide new insight into the role of lncRNAs and mRNAs, and would advance the understanding of lncRNA-mediated mechanisms in early development of fish.

Comparative microRNAs expression profiles analysis during embryonic development of common carp, Cyprinus carpio.

MicroRNAs (miRNAs) play important roles in biological processes by regulating specific gene expression. Limited miRNAs information is available on embryonic development in common carp (Cyprinus carpio) so far. In this study, six important embryonic development stages of C.carpio were collected to perform a times-series of small RNA-seq experiments from cleavage, blastocyst, gastrulation, organ formation, hatching stage to 1 day post-hatching larva. The expression profiles of miRNAs were identified and differentially expressed miRNAs (DEMs) were screened out based on pairwise comparison. A mean of 12,744,989 raw reads and 9,888,123 clean reads were obtained from each library. A total of 2565 miRNAs were identified. 68 of 204 DEMs were overlapped with stage-specific miRNAs, in which 15 were known miRNAs and seemed to play a key role in embryogenesis. Additionally, time-course expression reveals several intriguing fluctuations during embryogenesis. Numerous signaling pathways were identified in embryonic development, including the phototransduction, hippo signaling pathway, Wnt, melanogenesis, histidine metabolism and fatty acid biosynthesis. The results would provide new insight into the roles of miRNAs in embryonic development, and would help us to advance the understanding of miRNA-mediated mechanisms in embryonic development of fish.

Dynamic transcriptome sequencing and analysis during early development in the bighead carp (Hypophthalmichthys nobilis).

BACKGROUND: Early development is a key process of the life history of fish. However, the relationship between the transcriptome and the dynamic regulation of early development is still uncharacterized in the bighead carp (Hypophthalmichthys nobilis). In the present study, we performed transcriptome analysis of six development stages in H. nobilis, aiming to understand the dynamic molecular regulation of early development in this fish. RESULTS: A total of 76,573 unigenes were assembled from clean sequence reads, with an average length of 1768 base. Among which, 41,742 (54.54%) unigenes were annotated to public protein databases, and an additional 59,014 simple sequence repeat (SSR) loci were identified among the unigenes. Furthermore, 30,199 differentially expressed transcripts (DETs) (fold change > 4 or < 0.25, and the false discovery rate FDR < 0.01) were observed in comparisons between the adjacent developmental stages, and nine expression patterns (profiles) were simulated using series-cluster analysis across six developmental stages. The unigenes expression level markedly increased after the DS1 stage (early blastula), and the numbers of DETs gradually decreased during subsequent development. The largest transcriptomic change (up- or down-regulated) was detected during the period from DS1 to DS2 (6-somite stage), which was enriched for many biological processes and metabolic pathways related to maternal to zygotic transition (MZT). Distinctly protein-protein interaction (PPI) networks were plotted for DETs during the period from DS1 to DS2. The genes (or proteins) from the same pathways were integrated together, and showed with obvious co-regulation patterns. In the series-cluster analysis, a remarkable profile of gene expression (profile_48) was identified that is probably related to the hatching during H. nobilis development, and the strict co-expression of a hatching enzyme gene (hce1) with 33 other annotated genes was identified from this profile. CONCLUSIONS: The results indicated that strict dynamic regulation occurs during the early development in H. nobilis, especially in embryogenesis before hatching. This study provides valuable new information and transcriptomic resources related to H. nobilis early development, and for certain events such as MZT and hatching.

Characterization and functional analysis of slc7a11 gene, involved in skin color differentiation in the red tilapia.

Red tilapia has become more popular for aquaculture production in China in recent years. However, the pigmentation differentiation that has resulted from the process of genetic breeding and skin color variation during the overwintering period are the main problems limiting the development of commercial culture. The genetic basis of skin color differentiation is still not understood. Solute carrier family 7 member 11 (slc7a11) has been identified to be a critical genetic regulator of pheomelanin synthesis in the skin of mammals. However, little information is available about its molecular characteristics, expression, location and function in skin color differentiation of fish. In this study, three complete cDNA sequences (2159 bp, 2190 bp and 2249 bp) of slc7a11 were successfully isolated from Malaysian red tilapia, encoding polypeptides of 492, 525 and 492 amino acids respectively. Quantitative real-time PCR demonstrated that slc7a11 mRNA expression is high in the ventral skin of PR (pink with scattered red spots) fish. Immunofluorescence analysis revealed that xCT (the protein encoded by slc7a11) was concentrated mainly in the cytoplasm and nucleus of both the dorsal and ventral skin cells of fish. After RNA interference of slc7a11, slc7a11 and cbs mRNA expressions decreased, but the tyr mRNA expression increased in the skin of fish. Results suggest that slc7a11 plays an important role in skin color formation and differentiation of red tilapia through the melanogenesis pathway.

Long noncoding RNA and mRNA expression profiles following igf3 knockdown in common carp, Cyprinus carpio.

As a novel IGF system member, igf3 plays an important role in gonadal development of teleost fish. Although studies have reported the unusual expression of igf3 in fish gonad, whether the igf3 affects the expression of long noncoding RNAs (lncRNAs) in gonad remains unknown. In this study, an igf3 knockdown common carp (Cyprinus carpio) model was established by RNA interference. Then RNA sequencing of C. carpio gonad after igf3 knockdown was performed. A total of 327,169,410 and 306,305,018 clean reads were identified from control and igf3-dsRNA interference group, respectively. After a stringent filtering, RNA-seq yielded 14199 lncRNA and 106932 mRNA transcripts with 124 and 353 differentially expressed lncRNAs and mRNAs. Our dataset provides an extensive resource for understanding the potential regulatory molecular mechanism of igf3 in early stage of gonadal development in C. carpio.

Identification and characterization of skin color microRNAs in Koi carp (Cyprinus carpio L.) by Illumina sequencing.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, small (21-25 nucleotide), non-coding RNAs that play important roles in numerous biological processes. Koi carp exhibit diverse color patterns, making it an ideal subject for studying the genetics of pigmentation. However, the influence of miRNAs on skin color regulation and variation in Koi carp is poorly understood. RESULTS: Herein, we performed small RNA (sRNA) analysis of the three main skin colors in Koi carp by Illumina sequencing. The results revealed 330, 397, and 335 conserved miRNAs (belonging to 81 families) and 340, 353, and 351 candidate miRNAs in black, red, and white libraries, respectively. A total of 164 differentially expressed miRNAs (DEMs) and 14 overlapping DEMs were identified, including miR-196a, miR-125b, miR-202, miR-205-5p, miR-200b, and etc. Target prediction and functional analysis of color-related miRNAs such as miR-200b, miR-206, and miR-196a highlighted putative target genes, including Mitf, Mc1r, Foxd3, and Sox10 that are potentially related to pigmentation. Determination of reference miRNAs for relative quantification showed that let-7a was the most abundant single reference gene, and let-7a and miR-26b was the most abundant combination. CONCLUSIONS: The findings provide novel insight into the molecular mechanisms determining skin color differentiation in Koi carp, and serve as a valuable reference for future studies on tissue-specific miRNA abundance in Koi carp.

Erratum: Comparative Transcriptome Analysis Identifies Candidate Genes Related to Skin Color Differentiation in Red Tilapia.

This corrects the article DOI: 10.1038/srep31347.

Comparative microRNA-seq Analysis Depicts Candidate miRNAs Involved in Skin Color Differentiation in Red Tilapia.

Differentiation and variation in body color has been a growing limitation to the commercial value of red tilapia. Limited microRNA (miRNA) information is available on skin color differentiation and variation in fish so far. In this study, a high-throughput Illumina sequencing of sRNAs was conducted on three color varieties of red tilapia and 81,394,491 raw reads were generated. A total of 158 differentially expressed miRNAs (|log2(fold change)| ≥ 1 and q-value ≤ 0.001) were identified. Target prediction and functional analysis of color-related miRNAs showed that a variety of putative target genes—including slc7a11, mc1r and asip—played potential roles in pigmentation. Moreover; the miRNA-mRNA regulatory network was illustrated to elucidate the pigmentation differentiation, in which miR-138-5p and miR-722 were predicted to play important roles in regulating the pigmentation process. These results advance our understanding of the molecular mechanisms of skin pigmentation differentiation in red tilapia.