Lanmei Wang

To investigate the molecular mechanisms of muscle fiber development underlying locomotion differences between two common carp (Cyprinus carpio) strains cultured in a rice-fish symbiosis system, we conducted comparative transcriptomic and proteomic analyses of muscle tissues collected from indolent Congjiang (CJ) and active FFRC No. 2 (FR) strains cultured under identical paddy field conditions. RNA-seq revealed 3708 differentially expressed genes (DEGs), and proteomic analysis identified 108 differentially expressed proteins (DEPs). Functional enrichment and protein-protein interaction (PPI) network analyses highlighted key genes (psma3, ppargc1a, ube3a) and proteins (Myh, Ckmt2b, Suclg2) associated with proteasome activity, adipocytokine signaling, and ubiquitin-mediated protein degradation. Quantitative PCR (RT-qPCR) analysis of ubiquitin-proteasome pathway-related genes confirmed significantly higher expression levels in the muscle tissue of CJ compared with FR. After a 21-day interference experiment, ube3a knockdown increased average muscle fiber area and diameter and reduced muscle fiber density in juvenile CJ, indicating that ube3a knockdown might promote muscle development in common carp. Subsequent swimming performance measurements can be conducted to further explore the impact of ube3a on fish locomotion. These results provide molecular insights into fish locomotion capacity related to behavioral adaptation in agroecological systems.

Cold stress due to overwintering is considered a major issue limiting the development of the red tilapia ( Oreochromis spp.) industry. This study was the first to systematically investigate the effects of cold stress on skin color, immuno‐antioxidant function, muscle compositions, and intestinal microbiota of red tilapia during overwintering. Fish (initial weight: 72.71 ± 1.32 g) were divided into the cold stress group (cold) and the control group. In the control group, the water temperature (WT) was maintained at 20°C, which is consistent to with the overwintering WT indoors or greenhouse in the local area. In the cold group, the WT was decreased from 20°C to 8°C by 2°C per day during the experiment. At the end of the experiment, we found that the L ^∗ value of fish ventral skin in the cold group was lower than that in the control group ( p < 0.05). The superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and total antioxidant capacity (T‐AOC) activities were higher, but complement 3 (C3) and complement 4 (C4) activities were lower in the liver of cold group fish than those in the control group ( p < 0.05). For the gill tissue, the SOD, MDA, and GSH activities were higher, but the C3 activity was lower in the cold group than those in the control group ( p < 0.05). The total amino acid (TAA) content in the muscle of fish in the cold group was higher than that in the control group ( p < 0.05). 16 s rRNA analysis showed that the intestinal microbial abundance and alpha diversity of fish in the cold group decreased ( p < 0.05). Component analysis revealed a significantly higher relative abundance of Cetobacterium spp. in the fish of the cold group. The findings of this study will provide a valuable understanding of fish cold stress during overwintering, which will contribute to facilitating the breeding of new cold‐resistant varieties of red tilapia in the future.

Cold stress during overwintering is considered a bottleneck problem limiting the development of the red tilapia (Oreochromis spp.) industry, and the regulation mechanism is currently not well understood. In this study, the fish (initial weight: 72.71 ± 1.32 g) were divided into the cold stress group (cold) and the control (normal) group. In the control group, the water temperature was maintained at 20 °C, which is basically consistent with the overwintering water temperature in greenhouses of local areas. In the cold group, the water temperature decreased from 20 °C to 8 °C by 2 °C per day during the experiment. At the end of the experiment, the levels of fish serum urea nitrogen, glucose, norepinephrine, alkaline phosphatase, total bilirubin, and total cholesterol in the cold group changed significantly compared with that in the control group (P < 0.05). Then transcriptome sequencing and LC–MS metabolome of brain tissue were further employed to obtain the mRNA and metabolite datasets. We found that the FoxO signaling pathway and ABC transporters played an important role by transcriptome–metabolome association analysis. In the FoxO signaling pathway, the differentially expressed genes were related to cell cycle regulation, apoptosis and immune-regulation, and oxidative stress resistance and DNA repair. In the ABC transporters pathway, the ATP-binding cassette (ABC) subfamily abca, abcb, and abcc gene expression levels, and the deoxycytidine, L-lysine, L-glutamic acid, L-threonine, ornithine, and uridine metabolite contents changed. Our results suggested that the cold stress may promote apoptosis through regulation of the FoxO signaling pathway. The ABC transporters may respond to cold stress by regulating amino acid metabolism. The results provided a comprehensive understanding of fish cold stress during overwintering, which will facilitate the breeding of new cold-resistant varieties of red tilapia in the future.

The cold stress during overwintering is considered the bottleneck of red tilapia industry. In this study, the water temperature (WT) was reduced by 2 °C per day from 20 °C to 8 °C in the cold (C) group. Then transcriptome of brain(B), gill(G), liver(L) and skin(S) tissues and proteome of G, L and S tissues were performed in C and Normal (N) (WT: 20 °C) group. 24 transcriptomes were completed, and 168.8 Gb data were obtained, with more than 5.89 Gb clean data of each sample. A total of 30499 annotation results were obtained with 3199, 4697, 4393, and 3382 differentially expressed mRNAs in NB_vs_CB, NG_vs_CG, NL_vs_CL, NS_vs_CS. 18 DIA proteomes were performed, and 6341 proteins were obtained with 178, 500 and 166 differentially expressed proteins in NG_vs_CG, NL_vs_CL, NS_vs_CS. Our datasets can be reused for key genes and proteins identification, omics joint analysis and regulatory mechanism analysis of low temperature or cold stress in fish, which will help understanding the regulatory mechanism and facilitate the molecular selective breeding of cold-resistant varieties of fish.

Eriocheir sinensis is traditionally a native high-value crab that is widely distributed in eastern Asia, and the precocity is considered the bottleneck problem affecting the development of the industry. The precocious E. sinensis is defined as a crab that reaches complete sexual maturation during the first year of its lifespan rather than as normally in the second year. However, the exact regulatory mechanisms underlying the precocity are still unclear to date. This study is the first to explore the mechanism of precocity with transcriptome-metabolome association analysis between the precocious and normal sexually mature E. sinensis. Our results indicated that the phenylalanine metabolism (map00360) and neuroactive ligand-receptor interaction (map04080) pathways play an important role in the precocity in the ovary of E. sinensis. In map00360, the predicted aromatic-L-amino-acid decarboxylase and 4-hydroxyphenylpyruvate dioxygenase isoform X1 genes and the phenethylamine, phenylethyl alcohol, trans-2-hydroxycinnamate, and L-tyrosine metabolites were all down-regulated in the ovary of the precocious E. sinensis. The map04080 was the common KEGG pathway in the ovary and hepatopancreas between the precocious and normal crab. In the ovary, the predicted growth hormone secretagogue receptor type 1 gene was up-regulated, and the L-glutamate metabolite was down-regulated in the precocious E. sinensis. In the hepatopancreas, the predicted forkhead box protein I2 gene and taurine metabolite were up-regulated and the the L-glutamate metabolite was down-regulated in the precocious crab. There was no common pathway in the testis. Numerous common pathways in the hepatopancreas between male precocious and normal crab were identified. The specific amino acids, fatty acids and flavorful nucleotide (inosine monophosphate (MP), cytidine MP, adenosine MP, uridine MP, and guanosine MP) contents in the hepatopancreas and gonads further confirmed the above omics results. Our results suggest that the phenylalanine metabolism may affect the ovarian development by changing the contents of the neurotransmitter and tyrosine. The neuroactive ligand–receptor interaction pathway may affect the growth by changing the expressions of related genes and affect the umami taste of the gonads and hepatopancreas through the differences of L-glutamate metabolite in the precocious E. sinensis. The results provided valuable and novel insights on the precocious mechanism and may have a significant impact on the development of the E. sinensis aquaculture industry.

Skin color variation and differentiation are bottlenecks in the aquaculture industry of red tilapia. The mitfa gene primarily regulates melanocyte survival and proliferation in fish skin pigmentation. In this study, a recombinant expression vector for Mitfa expression was successfully constructed. After induction and purification, nearly 600 μg of Mitfa protein was purified from the fermentation supernatant. His pull-down assay, followed by LC-MS/MS analysis, revealed 10 proteins that interact with the Mitfa protein. Five of these proteins were annotated in the melanogenesis and tyrosine metabolism pathways, including Krt, Tpm, Myh, Tyr and Dct. The purified Mitfa recombinant protein was injected into red tilapia, and its activity and function were examined. The expression levels of the mitfa, tyr, tyrp1, and dct genes increased greatly in the dorsal skin after injection (p < 0.05); meanwhile, the melanin content and tyrosinase activity increased significantly (p < 0.05). These findings provide a theoretical basis for comprehending the function and regulatory mechanism of the red tilapia mitfa gene.

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.). 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. 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.

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. 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. 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.

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.