High-temperature stress response: Insights into the molecular regulation of American shad (Alosa sapidissima) using a multi-omics approach.

High temperature is an important abiotic stressor that limits the survival and growth of aquatic organisms. American shad (Alosa sapidissima), a migratory fish suitable for culturing at low temperatures, is known for its delicious taste and thus has high economic value. Studies concerning changes in A. sapidissima under high temperature are limited, especially at the gene expression and protein levels. High-temperature stress significantly reduced the survival rates and increased vacuolar degeneration and inflammatory infiltration in the gills and liver. High temperature increased the activities of SOD, CAT, and cortisol, with a trend of initial increase followed by decreases in MDA, ALP, and LDH, and irregular changes in T-AOC and Na-K-ATPase. Comprehensive analysis of the transcriptome, proteome, and metabolome of gills from fish treated with different culture temperatures (24, 27, and 30 °C) revealed that differentially expressed genes, proteins, and metabolites were highly enriched in pathways involved in protein digestion and absorption, protein processing in endoplasmic reticulum, metabolic pathways, and purine metabolism. Gene expression and protein profiles indicated that genes coding for antioxidants (i.e., cat and alpl) and members of the heat shock protein (i.e., HSP70, HSP90AA1, and HSP5) were significantly upregulated. Additionally, a conjoint analysis revealed that several key enzymes, including nucleoside diphosphate kinase 2, adenosine deaminase, and ectonucleoside triphosphate diphosphohydrolase 5/6 were altered, thereby affecting the metabolism of guanosine, guanine, and inosine. An interaction network further confirmed that levels of the essential amino acids DL-arginine and L-histidine were significantly reduced, and corticosterone levels were significantly increased, suggesting that A. sapidissima may be more dependent on amino acids for energy in vivo. Overall, this work suggests that living in a high-temperature environment leads to differential defense responses in fishes. The results provide novel perspectives for studying the molecular basis of adaptation to climate change in A. sapidissima and for genetic selection.

Research on main influencing factors and complete support technology for dynamic pressure and large deformation roadway.

To determine the main factors influencing dynamic pressure and large deformation roadways, a targeted set of support technologies was designed. The 2603 air inlet roadway of the Zhangcun coal mine in Lu'an, Shanxi Province, was taken as an example. The influence of the Wenwangshan South normal fault and in situ stress field on the dynamic pressure roadway was analyzed theoretically, and the main factors influencing this dynamic pressure and large deformation roadway under natural geological conditions were determined. The effect of the existing roadway support scheme was evaluated by field test methods such as nondestructive bolt testing. The influence of mining two working faces on the dynamic pressure and large deformation roadway was studied by the FLAC3D numerical simulation method. On this basis, a new grouting material was developed, a complete set of technical schemes of full-section integrated cooperative support of dynamic pressure and large deformation roadways was proposed, and the field application effect was verified. The results showed that under natural geological conditions, the 2603 air inlet roadway was located within the influence range of the Wenwangshan South normal fault, which was significantly affected and controlled by the fault. The included angle between the roadway extension direction and the maximum principal stress was 74°, which was not conducive to the stability of the roadway. The range of the roadway loose zone was large. Under the existing support conditions, the surrounding rock could not form a relatively stable structure, which was one of the main reasons for the large deformation of the surrounding rock in the dynamic pressure roadway. The 2603 air inlet roadway was affected by the mining of both the adjacent working face and the 2603 working face. The stresses were superimposed, and the roadway was greatly deformed and damaged. A new grouting material was developed. A crosslinking agent prepared by toluene diisocyanate and polyether polyol was added to the existing polyurethane material to form a new grouting material, and a complete supporting technical scheme was proposed. The field application results showed that the displacement and floor heave of both sides of the roadway were reduced by approximately 87%, the deformation and failure of the coal and rock mass of the roadway were effectively controlled, and the deformation of the dynamic pressure roadway was greatly reduced.

microRNA regulation of skin pigmentation in golden-back mutant of crucian carp from a rice-fish integrated farming system.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small non-coding RNAs (21-25 nucleotides) that act as essential components of several biological processes. Golden-back crucian carp (GBCrC, Carassius auratus) is a naturally mutant species of carp that has two distinct body skin color types (golden and greenish-grey), making it an excellent model for research on the genetic basis of pigmentation. Here, we performed small RNA (sRNA) analysis on the two different skin colors via Illumina sequencing. RESULTS: A total of 679 known miRNAs and 254 novel miRNAs were identified, of which 32 were detected as miRNAs with significant differential expression (DEMs). 23,577 genes were projected to be the targets of 32 DEMs, primarily those involved in melanogenesis, adrenergic signaling in cardiomyocytes, MAPK signaling pathway and wnt signaling pathway by functional enrichment. Furthermore, we built an interaction module of mRNAs, proteins and miRNAs based on 10 up-regulated and 13 down-regulated miRNAs in golden skin. In addition to transcriptional destabilization and translational suppression, we discovered that miRNAs and their target genes were expressed in the same trend at both the transcriptional and translational levels. Finally, we discovered that miR-196d could be indirectly implicated in regulating melanocyte synthesis and motility in the skin by targeting to myh7 (myosin-7) gene through the luciferase reporter assay, antagomir silencing in vivo and qRT-PCR techniques. CONCLUSIONS: Our study gives a systematic examination of the miRNA profiles expressed in the skin of GBCrC, assisting in the comprehension of the intricate molecular regulation of body color polymorphism and providing insights for C. auratus breeding research.

Supporting optimization of thick seam roadway with top coal based on orthogonal matrix analysis.

Aiming at the problem of large deformation and difficulty in surrounding rock control of the top coal roadway in thick seam, theoretical analysis, theoretical analysis, numerical simulation, orthogonal matrix analysis and other methods were used to study the roof deformation and support parameter optimization of the top coal roadway in thick seam. Firstly, the structural model and roof mechanical model of the top coal roadway in thick seam were established, and the deformation coefficient TK was defined based on the relationship between curvature radius and bending moment, maximum bending moment and ultimate tensile strength of beam. According to the ratio of deformation rate between TK and beam to determine the roof deformation mode of top coal roadway, the discriminant conditions of roadway roof stability under two deformation conditions are obtained. Due to the characteristics of serious coal-rock fragmentation, large roof deformation, and integration of top coal and side coal. Therefore, the combined support method of "high prestressed long and short anchor cables" is proposed by double arch bearing structure control technology. Finally, based on the orthogonal matrix analysis method of supporting parameters optimization of the top coal roadway in thick seam, the analysis amount of supporting scheme is significantly reduced, the comprehensive evaluation of multi-factor and multi-supporting effect of roadway support is realized, and the optimal supporting scheme is obtained. Compared with the surrounding rock of the roadway without support, the deformation of the roof is reduced by 27.27%, the deformation of the two sides is reduced by 45.24%, and the tensile failure volume is reduced by 54.66%. The top coal roadway in thick seam has been effectively controlled, which provides guarantee for high yield and high efficiency of the mine.

Bolt-Cable-Mesh Integrated Support Technology for Water Drenching Roadway in Thick Coal Seam.

Aiming at the problems of large deformation and difficult control of the mining roadway water drenching in a thick coal seam, the principle of double-arch zoning cooperative surrounding rock control is studied by using the combined method of theoretical analysis, numerical simulation, and industrial experiment. The water-rock interaction of the surrounding rock of water-drenching roadway is proposed, taking into account the damage to the mechanical parameters of the surrounding rock caused by soaking water. The water-mechanical-damage coupling model for surrounding rock of a coal seam roadway is constructed, and is numerically solved by the code development using the FISH language. The principle of "high prestressed bolt-cable-mesh" zone coordinated control is revealed, and the existence of a compressive stress arch is verified by FLAC3D software. The three support schemes were compared and analyzed by numerical simulation software. We affirmed the bolt length of 2400 mm, spacing of 0.9 m, row spacing of 1 m, cable length of 7300 mm, and arrangement 3-2-3 as the optimal support scheme and applied this scheme in the Zhangcun Coal Mine. The results show that the maximum deformation of the roadway roof is 142 mm, and the ultimate convergence of the two sides is 83 mm. The surrounding rock has been effectively controlled.

Studies on the molecular level changes and potential resistance mechanism of Coreius guichenoti under temperature stimulation.

In this study, we used transcriptome and proteome technology to analyze molecular level changes in tissues of Coreius guichenoti cultured at high temperature (HT) and low temperature (LT). We also screened for specific anti-stress genes and proteins and evaluated the relationships between them. We identified 201,803 unigenes and 10,623 proteins. Compared with the normal temperature (NT), 408 genes and 1,204 proteins were up- or down-regulated in brain tissues, respectively, at HT, and the numbers were 8 and 149 at LT. In gill tissues, the numbers were 101 and 1,745 at HT and 27 and 511 at LT. In gill tissues at both temperatures, the degree of down-regulation (average, HT 204.67-fold, LT 443.13-fold) was much greater than that of up-regulation (average, HT 28.69-fold, LT 17.68-fold). The protein expression in brain (average, up 52.67-fold, down 13.54-fold) and gill (average, up 73.02-fold, down 12.92-fold) tissues increased more at HT than at LT. The protein expression in brain (up 3.77-fold, down 4.79-fold) tissues decreased more at LT than at HT, whereas the protein expression in gill (up 8.64-fold, down 4.35-fold) tissues was up-regulated more at LT than at HT. At HT, brain tissues were mainly enriched in pathways related to metabolism and DNA repair; at LT, they were mainly enriched in cancer-related pathways. At both temperatures, gill tissues were mainly enriched in pathways related to cell proliferation, apoptosis, immunity, and inflammation. Additionally, Kyoto Encyclopedia of Genes and Genomes pathway analysis showed more differentially expressed proteins in gill tissues than in brain tissues at HT and LT, and temperature stimulation led to the strengthening of metabolic pathways in both tissues. Of the 96 genes we identified as potentially being highly related to temperature stress (59 from transcriptome and 38 from proteome data), we detected heat shock protein 70 in both the transcriptome and proteome. Our results improved our understanding of the differential relationship between gene expression and protein expression in C. guichenoti. Identifying important temperature stress genes will help lay a foundation for cultivating C. guichenoti, and even other fish species, that are resistant to HT or LT.

Molecular and functional analysis of the microphthalmia-associated transcription factor (mitf) gene duplicates in red tilapia.

In vertebrates, the microphthalmia-associated transcription factor (mitf) is at the hub of the melanin synthesis regulation network. However, little information is known about its molecular characterization, expression, location, or function in skin color differentiation and variation of red tilapia. The full-length cDNA sequences (1977 bp and 1999 bp) of mitfa and mitfb, encoding polypeptides of 491 and 514 amino acids, were effectively identified from red tilapia in this study. The Mitfa and Mitfb sequences of red tilapia clustered first with O. aureus, then with other teleost fish, according to phylogenetic analysis. Mitfa and mitfb mRNA were highly expressed in the brain, dorsal skin and eye tissues using quantitative real-time PCR. The mRNA expressions of mitfa and mitfb were the highest in the cleavage stage during the early development of red tilapia. Among three different colors of red tilapia, the expression levels of mitfa and mitfb were highest in the PB (pink with scattered black spots) dorsal skin. After overwintering, the mitfa and mitfb mRNA expressions were high in the dorsal skin of PB (color changed from pink to black). Mitfa and mitfb were mostly found in the epidermal layer of the dorsal skin, according to in situ hybridization (ISH) analysis. After injecting mitf-dsRNA duplicates along the tail vein of red tilapia, the activity of tyrosinase and the level of melanin in the dorsal skin both decreased significantly. The mRNA expressions of mitfa and its downstream genes (tyrb, tyrp1a and dct) decreased, whereas the mRNA expression of mitfb increased after mitfa-dsRNA injection. The mRNA expressions of mitfb, tyrb, tyrp1a and dct decreased, whereas the mRNA expression of mitfa increased after injecting mitfb-dsRNA. These findings suggest that mitf gene duplicates may play an important role in red tilapia skin color differentiation and variation via the melanogenesis pathway.

Transcriptome analysis of skin color variation during and after overwintering of Malaysian red tilapia.

The commercial value of red tilapia is hampered by variations in skin color during overwintering. In this study, three types of skin of red tilapia, including the skin remained pink color during and after overwintering (P), the skin changed from pink color to black color during overwintering and remained black color after overwintering (P-B), and the skin changed from pink color to black color during overwintering but recovered to pink color when the temperature rose after overwintering (P-B-P), were used to analyze their molecular mechanisms of color variation. The transcriptome results revealed that the P, P-B, and P-B-P libraries had 43, 42, and 43 million clean reads, respectively. The top 10 abundance mRNAs and specific mRNAs (specificity measure SPM > 0.9) were screened. After comparing intergroup gene expression levels, there were 2528, 1924, and 1939 differentially expressed genes (DEGs) between P-B-P and P-B, P-B-P and P, and P-B and P, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of color-related mRNAs showed that a number of DEGs, including tyrp1, tyr, pmel, mitf, mc1r, asip, tat, hpdb, and foxd3, might play a potential role in pigmentation. Additionally, the co-expression patterns of genes were detected within the pigment-related pathways by the PPI network from P-B vs. P group. Furthermore, DEGs from the apoptosis and autophagy pathways, such as baxα, beclin1, and atg7, might be involved in the fading of red tilapia melanocytes. The findings will aid in understanding the molecular mechanism underlying skin color variation in red tilapia during and after overwintering as well as lay a foundation for future research aimed at improving red tilapia skin color characteristics.

Comparative analysis of the gut microbiota in bighead carp under different culture patterns.

AIMS: To investigate the phylogenetic composition and functional potential of bighead carp (Hypophthalmichthys nobilis) gut microbiome in two rearing patterns (bighead carp polycultured with Oreochromis niloticus in pond A and bighead carp polycultured with Cyprinus carpio in pond B, respectively), as well as the changes of plankton in the cultured water at four different time points. METHODS AND RESULTS: The intestinal contents were sequenced using Illumina HiSeq of bacterial 16S rRNA. Cyanophyta and Chlorophyta were the prevalent phytoplankton in the water, whereas Rotifers and Protozoa were the predominant zooplankton. In all, 779,563 quality-filtered sequences and 8870 amplicon sequence variants were obtained from 24 samples that numbered T1A1 to T4A3 and T1B1 to T4B3, resulting in 35 phyla, with Proteobacteria, Firmicutes, Fusobacteria and Cyanobacteria dominating. According to alpha diversity and beta diversity measurements, the bacterial communities were diverse, Chao1 richness and Pielou's evenness were significantly lower in the T2B and T4B groups. The gut bacterial communities of T1A, T1B, T2A and T2B groups differed from those of other samples, which formed distinctly clusters with principal coordinate analysis and non-metric multidimensional scaling analysis. PICRUSt2 predictive function analysis revealed that different culture patterns influenced the gut microbiota metabolic capacity. CONCLUSIONS: Intestinal bacteria belonging to the phyla Proteobacteria, Firmicutes, Cyanobacteria and Fusobacteria are better suited to inhabit in various environments and perform specific functions. Furthermore, contact with the external environment and nutrient intake also stimulate the variety of intestinal microbiotas in polycultured bighead carp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comprehensive, high-throughput investigation of gut microbiota diversity in bighead carp during various seasons in two polycultured patterns and provide preliminary information on gut microbiome composition and changes, laying a crucial foundation for future research on fish culture patterns in various environments.

Dynamic Expression and Gene Regulation of MicroRNAs During Bighead Carp (Hypophthalmichthys nobilis) Early Development.

The early development of fish is regulated through dynamic and complex mechanisms involving the regulation of various genes. Many genes are subjected to post-transcriptional regulation by microRNAs (miRNAs). In the Chinese aquaculture industry, the native species bighead carp (Hypophthalmichthys nobilis) is important. However, the genetic regulation related to the early development of bighead carp is unknown. Here, we generated developmental profiles by miRNA sequencing to study the dynamic regulation of miRNAs during bighead carp early development. This study identified 1 046 miRNAs, comprising 312 known miRNAs and 734 uncharacterized miRNAs. Changes in miRNA expression were identified in the six early development stages. An obviously increased expression trend was detected during the development process, with the main burst of activity occurring after the earliest stage (early blastula, DS1). Investigations revealed that several miRNAs were dominantly expressed during the development process, especially in the later stages (e.g., miR-10b-5p, miR-21, miR-92a-3p, miR-206-3p, and miR-430a-3p), suggesting that these miRNAs exerted important functions during embryonic development. The differentially expressed miRNAs (DEMs) and time-serial analysis (profiles) of DEMs were analyzed. A total of 372 miRNAs were identified as DEMs (fold-change >2, and false discovery rate <0.05), and three expression profiles of the DEMs were detected to have co-expression patterns (r > 0.7, and p < 0.05). The broad negative regulation of target genes by miRNAs was speculated, and many development-related biological processes and pathways were enriched for the targets of the DEMs, which might be associated with maternal genome degradation and embryogenesis processes. In conclusion, we revealed the repertoire of miRNAs that are active during early development of bighead carp. These findings will increase our understanding of the regulatory mechanisms of early development of fish.

Evolution and genetics of bighead and silver carps: Native population conservation versus invasive species control.

Bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix), collectively called bigheaded carps, are cyprinids native mainly to China and have been introduced to over 70 countries. Paleontological and molecular phylogenetic analyses demonstrate bighead and silver carps originated from the Yangtze-Huanghe River basins and modern populations may have derived from the secondary contact of geographically isolated fish during the last glacial events. Significant genetic differences are found among populations of native rivers (Yangtze, Pearl, and Amur) as well as introduced/invasive environments (Mississippi R., USA and Danube R., Hungary), suggesting genetic backgrounds and ecological selection may play a role in population differentiation. Population divergence of bighead carp or silver carp has occurred within their native rivers, whereas, within the Mississippi River Basin (MRB)-an introduced region, such genetic differentiation is likely taking place at least in silver carp. Interspecific hybridization between silver and bighead carps is rare within their native regions; however, extensive hybridization is observed in the MRB, which could be contributed by a shift to a more homogenous environment that lacks reproductive isolation barriers for the restriction of gene flow between species. The wild populations of native bighead and silver carps have experienced dramatic declines; in contrast, the introduced bigheaded carps overpopulate the MRB and are considered two invasive species, which strongly suggests fishing capacity (overfishing and underfishing) be a decisive factor for fishery resource exploitation and management. This review provides not only a global perspective of evolutionary history and population divergence of bigheaded carps but also a forum that calls for international research collaborations to deal with critical issues related to native population conservation and invasive species control.

MicroRNA-206 Regulation of Skin Pigmentation in Koi Carp (Cyprinus carpio L.).

MicroRNAs (miRNAs) are ∼22 nucleotide non-coding RNA molecules that act as crucial roles in plenty of biological processes. However, the molecular and cellular mechanisms of miRNAs to regulate skin color differentiation and pigmentation in fish have not been fully understood. Herein, we revealed that miR-206, a skin-enriched miRNA, regulates melanocortin 1 receptor (Mc1r, a key regulator of melanogenesis) expression by binding to its 3'-untranslated (UTR) region through bioinformatics and luciferase reporter assay in koi carp (Cyprinus carpio L.). The analysis of spatial and temporal expression patterns suggested that miR-206 is a potential regulator in the skin pigmentation process. Then, we silenced it in vivo with an antagomir method. The result showed a substantial increase of Mc1r mRNA expression and protein level, and also its downstream genes: tyrosinase (Tyr) and dopachrome tautomerase (Dct) that encoding key enzymes involved in melanin synthesis. Moreover, we constructed the miRNA-206 sponge lentivirus vector to transfect koi carp melanocytes in vitro, further checked the functions of melanocytes using Cck-8 and Transwell assays. As a result, inhibition of miR-206 significantly up-regulated Mc1r mRNA expression and protein level and accelerated the melanocyte proliferation and migration ability compared with the scrambled-sequence negative control group (miR-NC). Overall, these findings provide the evidence that miR-206 plays a regulatory role in the skin color pigmentation through targeting the Mc1r gene and would facilitate understanding the molecular regulatory mechanisms underlying miRNA-mediated skin color pigmentation in koi carp.

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.

Integrated analysis of long non-coding RNA and mRNA expression in different colored skin of koi carp.

BACKGROUND: Long non-coding RNAs (lncRNAs) perform crucial roles in biological process involving complex mechanisms. However, information regarding their abundance, characteristics and potential functions linked to fish skin color is limited. Herein, Illumina sequencing and bioinformatics were conducted on black, white, and red skin of Koi carp (Cyprinus carpio L.). RESULTS: A total of 590,415,050 clean reads, 446,614 putative transcripts, 4252 known and 72,907 novel lncRNAs were simultaneously obtained, including 92 significant differentially expressed lncRNAs and 722 mRNAs. Ccr_lnc5622441 and Ccr_lnc765201 were up-regulated in black and red skin, Ccr_lnc14074601 and Ccr_lnc2382951 were up-regulated in white skin, and premelanosome protein a (Pmela), Pmelb and tyrosinase (Tyr) were up-regulated in black skin. The expression patterns of 18 randomly selected differentially expressed genes were validated using the quantitative real-time PCR method. Moreover, 70 lncRNAs acting on 107 target mRNAs in cis and 79 lncRNAs acting on 41,625 target mRNAs in trans were investigated. The resulting co-expression networks revealed that a single lncRNA can connect with numerous mRNAs, and vice versa. To further reveal their potential functions, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed, and membrane, pigment cell development, cAMP signaling, melanogenesis and tyrosine metabolism appear to affect skin pigmentation. Additionally, three lncRNAs (Ccr_lnc142711, Ccr_lnc17214525 and Ccr_lnc14830101) and three mRNAs (Asip, Mitf and Tyr) involved in the melanogenesis pathway were investigated in terms of potential functions in embryogenesis and different tissues. CONCLUSIONS: The findings broaden our understanding of lncRNAs and skin color genetics, and provide new insight into the mechanisms underlying lncRNA-mediated pigmentation and differentiation in Koi carp.

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.