Non-invasive methods for heart rate measurement in fish based on photoplethysmography.

Heart rate is a crucial physiological indicator for fish, but current measurement methods are often invasive or require delicate manipulation. In this study, we introduced two non-invasive and easy-to-operate methods based on photoplethysmography, namely reflectance-type photoplethysmography (PPG) and remote photoplethysmography (rPPG), which we applied to the large yellow croaker (Larimichthys crocea). PPG showed perfect synchronization with electrocardiogram (ECG), with a Pearson's correlation coefficient of 0.99999. For rPPG, the results showed good agreement with ECG. Under active provision of green light, the Pearson's correlation coefficient was 0.966, surpassing the value of 0.947 under natural light. Additionally, the root mean square error was 0.810, which was lower than the value of 1.30 under natural light, indicating not only that the rPPG method had relatively high accuracy but also that green light may have the potential to further improve its accuracy.

Machine vision-assisted genomic prediction and genome-wide association of spleen-related traits in large yellow croaker infected with visceral white-nodules disease.

High-resolution and high-throughput genotype-to-phenotype studies in fish are rapidly advancing, driven by innovative technologies that aim to address the challenges of modern breeding models. In recent years, machine vision and deep learning techniques, particularly convolutional neural networks (CNNs), have achieved significant success in image recognition and segmentation. Moreover, qualitative and quantitative analysis of disease resistance has always been a crucial field of research in genetics. This motivation has led us to investigate the potential of large yellow croaker visceral white-nodules disease (VWND) in encoding information on disease resistance for the task of accession classification. In this study, we proposed an image segmentation framework for the feature extraction of the spleen after VWND infection based on machine vision. We utilized deep CNNs and threshold segmentation for automatic feature learning and object segmentation. This approach eliminates subjectivity and enhances work efficiency compared to using hand-crafted features. Additionally, we employed spleen-related traits to conduct genome-wide association analysis (GWAS), which led to the identification of 24 significant SNPs and 10 major quantitative trait loci. The results of function enrichment analysis on candidate genes also indicated potential relationships with immune regulation mechanisms. Furthermore, we explored the use of genomic selection (GS) technology for phenotype prediction of extreme individuals, which further supports the predictability of spleen-related phenotypes for VWND resistance in large yellow croakers. Our findings demonstrate that artificial intelligence (AI)-based phenotyping approaches can deliver state-of-the-art performance for genetics research. We hope this work will provide a paradigm for applying deep learning and machine vision to phenotyping in aquaculture species.

Integrated mRNA and miRNA expression analyses for Cryptocaryon irritans resistance in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. However, cryptocaryonosis caused by Cryptocryon irritans infection has brought huge economic losses and threatened the healthy and sustainable development of L. crocea industry. Recently, a new C. irritans resistance strain of L. crocea (RS) has been bred using genomic selection technology in our laboratory work. However, the molecular mechanisms for C. irritans resistance of RS have not been fully understood. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that are post-transcriptional regulators, and they play vital roles in immune process of bony fish. Identification of anti-C.irritans relevant miRNA signatures could, therefore, be of tremendous translational value. In the present study, integrated mRNA and miRNA expression analysis was used to explore C. irritans resistance mechanisms of the L. crocea. RS as well as a control strain (CS) of L. crocea, were artificially infected with C. irritans for 100 h, and their gill was collected at 0 h (pre-infection), 24 h (initial infection), and 72 h (peak infection) time points. The total RNA from gill tissues was extracted and used for transcriptome sequencing and small RNA sequencing. After sequencing, 23,172 known mRNAs and 289 known miRNAs were identified. The differential expression was analyzed in these mRNAs and mRNAs and the interactions of miRNA-mRNA pairs were constructed. KEGG pathway enrichment analyses showed that these putative target mRNAs of differentially expressed miRNAs (DEMs) were enriched in different immune-related pathways after C. irritans infection in RS and CS. Among them, necroptosis was the immune-related pathway that was only significantly enriched at two infection stages of RS group (RS-24 h/RS-0h and RS-72 h/RS-0h). Further investigation indicates that necroptosis may be activated by DEMs such as miR-133a-3p, miR-142a-3p and miR-135c, this promotes inflammation responses and pathogen elimination. These DEMs were selected as miRNAs that could potentially regulate the C. irritans resistance of L. crocea. Though these inferences need to be further verified, these findings will be helpful for the research of the molecular mechanism of C. irritans resistance of L. crocea and miRNA-assisted molecular breeding of aquatic animals.

Comparative transcriptome analysis reveals immunoregulation mechanism of lncRNA-mRNA in gill and skin of large yellow croaker (Larimichthys crocea) in response to Cryptocaryon irritans infection.

BACKGROUND: Cryptocaryonosis caused by Cryptocaryon irritans is one of the major diseases of large yellow croaker (Larimichthys crocea), which lead to massive economic losses annually to the aquaculture industry of L. crocea. Although there have been some studies on the pathogenesis for cryptocaryonosis, little is known about the innate defense mechanism of different immune organs of large yellow croaker. RESULTS: In order to analyze the roles of long non-coding RNAs and genes specifically expressed between immune organs during the infection of C. irritans, in this study, by comparing transcriptome data from different tissues of L. crocea, we identified tissue-specific transcripts in the gills and skin, including 507 DE lncRNAs and 1592 DEGs identified in the gills, and 110 DE lncRNAs and 1160 DEGs identified in the skin. Furthermore, we constructed transcriptome co-expression profiles of L. crocea gill and skin, including 7,503 long noncoding RNAs (lncRNAs) and 23,172 protein-coding genes. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DEGs and the target genes of the DE lncRNAs in the gill were specifically enriched in several pathways related to immune such as HIF-1 signaling pathway. The target genes of DE lncRNAs and DEGs in the skin are specifically enriched in the complement and coagulation cascade pathways. Protein-protein interaction (PPI) network analysis identified 3 hub genes including NFKBIA, TNFAIP3 and CEBPB, and 5 important DE lncRNAs including MSTRG.24134.4, MSTRG.3038.5, MSTRG.27019.3, MSTRG.26559.1, and MSTRG.10983.1. The expression patterns of 6 randomly selected differentially expressed immune-related genes were validated using the quantitative real-time PCR method. CONCLUSIONS: In short, our study is helpful to explore the potential interplay between lncRNAs and protein coding genes in different tissues of L. crocea post C. irritans and the molecular mechanism of pathogenesis for cryptocaryonosis. HIGHLIGHTS: Skin and gills are important sources of pro-inflammatory molecules, and their gene expression patterns are tissue-specific after C. irritans infection. 15 DEGs and 5 DE lncRNAs were identified as hub regulatory elements after C. irritans infection The HIF-1 signaling pathway and the complement and coagulation cascade pathway may be key tissue-specific regulatory pathways in gills and skin, respectively.

Integrative analysis of GWAS and transcriptome reveals p53 signaling pathway mediates resistance to visceral white-nodules disease in large yellow croaker.

Visceral white-nodules disease (VWND), caused by Pseudomonas plecoglossicida, is one of the primary causes of morbidity and mortality in large yellow croaker aquaculture. Host disease resistance is a heritable trait that involves complex regulatory processes. However, the regulatory mechanism of bacterial resistance in large yellow croaker is still unclear. This study attempted to systematically evaluate the major genetic loci and transcriptional regulatory mechanisms associated with the resistance to VWND in large yellow croaker by crossover method studies. A large population of large yellow croaker was challenged with P. plecoglossicida, with survival time recorded and samples were taken for genotyping. Meanwhile, spleen samples that were used for RNA-seq to compare their transcriptomic profiles before and after infection were taken from resistant populations (RS) and susceptible control populations (CS) bred using the genomic selection (GS) technique. Genome-wide association analyses using 46 K imputed SNP genotypes highlighted that resistance is a polygenic trait. The integrative analysis results show the co-localization of the cd82a gene between disease resistance-related genetic loci and comparative transcriptional analysis. And functional enrichment analysis showed differential enrichment of the p53 signaling pathway in RS and CS groups, suggesting that there may be cd82a-mediated p53 signaling pathway activation for VWND resistance. This large-scale study provides further evidence for the heritability and transcriptional regulatory mechanisms of host inheritance of VWND resistance.

Transcriptome analysis reveals the temporal gene expression patterns in skin of large yellow croaker (Larimichthys crocea) in response to Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. In the past decades, cryptocaryonosis caused by Cryptocryon irritans has led to huge economic losses, posing great threat to the healthy and sustainable development of L. crocea mariculture industry. As the largest immunologically active mucosal organ in fish, skin provides the first defense line against external pathogens. To better understand the gene expression dynamics, the large yellow croakers were artificially infected with C. irritans and their skin tissues were collected at 0 h, 24 h, 48 h, 72 h and 96 h post infection. The total RNA in the skin tissues were extracted and the transcriptome were sequenced. After sequencing, a total of 1,131, 311, 140 million high quality RNA-seq reads were collected. A set of 215, 473, 968, 1055 differentially expressed genes were identified at 24 h, 48 h, 72 h and 96 h post infection respectively. Further analysis clustered these DEGs into six profiles and 75 hub genes for six profiles were identified. Among these hub genes, 18 immune related genes including TLR5, TOPK, NFKBIZ, MAPK14A were identified post C. irritans infection. Cytokine-cytokine receptor interaction was the only pathway that significantly enriched at four timepoints post infection. This study provides an in-depth understanding of skin transcriptome variance of large yellow croaker after C. irritans infection, which would be helpful for further understanding of the molecular mechanism of L. crocea in response to C. irritans infection.

Molecular mechanisms of an antimicrobial peptide piscidin (Lc-pis) in a parasitic protozoan, Cryptocaryon irritans.

BACKGROUND: Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture fish species and cause high lethality and economic loss. Current methods of controlling this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. Piscidins with broad spectrum antibacterial, antifungal and antiviral activities were found to have potent activity against C. irritans. Little, however, has been understood about the killing mechanisms of piscidins in parasites. RESULTS: In total, 57.12, 50.44, 55.86 and 47.87 million raw reads were generated from untreated theront and trophont, and piscidin (Lc-pis) treated theront and trophont libraries, respectively. After de novo assembly, 966,609 unigenes were generated with an average length of 420 bp: among these, 618,629 unigenes showed identity with sequences in one or more databases, with some showing to be significantly manipulated by Lc-pis treatment. The species classification showed that more than 25.8% unigenes from trophonts were homologous to the large yellow croaker (Larimichthys crocea) and less than 3.8% unigenes from theronts were matched. The homologous unigenes demonstrated that the tissue from host could exist in trophonts and might be transported to parasite via vesicular transports. Our analysis showed that regulatory transcripts were involved in vesicular trafficking. Among transcripts induced by Lc-pis, most genes up-regulated in treated and untreated theronts were involved in cell migration and apoptosis related pathways. Few transcripts were found to be down-regulated in treated and untreated trophonts related to cell structure and migration after treatment. CONCLUSIONS: This is the first transcriptome analysis of C. irritans exposed to Lc-pis, which enhanced the genomic resources and provided novel insights into molecular mechanisms of ciliates treated by cationic antimicrobial peptide. Our comprehensive transcriptome analysis can facilitate the identification of potential drug targets and vaccines candidates for controlling this devastating fish pathogen.

A novel stimulator of interferon gene (STING) from Larimichthys crocea and their involvement in immune response to ectoparasite Cryptocaryon irritans infection.

The marine white spot disease caused by protozoan ectoparasite Cryptocaryon irritans is a severe problem to the large yellow croaker farming industry. To understand the molecular immune mechanisms and improve its immune capacity are particular important. STING, one of the important second messengers in innate immune response process, plays pivotal roles in defensing against different pathogenic microorganisms. Many reports have pointed that STING could not only combine the uncovered dsDNA, ssDNA directly in the cytoplasm from the pathogens or biology itself, but it also could recognize cyclic diguanylate monophosphate (c-di-GMP), cyclic diadenylate monophosphate (c-di-AMP). Based on the STING sequence, a variant of the L. crocea STING (termed LcSTING2) was found by accident. RACE was used to clone the full-length cDNA of LcSTING2 which contained a 5'- UTR of 154 bp, a 3'-UTR of 592 bp and an ORF of 1227 bp encoding 408 amino acids. The predicted protein molecular weight (Mw) was 45.83 KDa and the estimated theoretical isoelectric point (pI) was 6.24. The deduced protein of LcSTING2 contains no signal peptide. One transmembrane motif (TM) in the N-terminal region, a TMEM173 domain and five putative motifs (RXR) found in resident endoplasmic reticulum proteins were also conserved. According to the partial genomic sequence, the unknown sequences were amplified, it contained 6 exons and 5 introns, and all the exon-intron boundaries were in accordance with classical GT-AG rule (GT/intron/AG). The similarity shared with fishes was higher than other high vertebrates. qRT-PCR results showed that LcSTING (two variants) distributed in all examined tissues, and it was the most abundant in gill. After challenged by C. irritans, LcSTING mRNA only appeared instantaneous up-regulation during the infective stage of theronts in the head kidney and was also up-regulated during the whole infectious cycle of C. irritans in the liver, which implied LcSTING gene was likely to be involved in the defensing against C. irritans infection, it is the first time to explore the STING taking part in the immune response to ectoparasite rather than bacterium or viruses.

Transcriptome analysis of the Larimichthys crocea liver in response to Cryptocaryon irritans.

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics.

Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion.

Stress, antioxidant defence and mucosal immune responses of the large yellow croaker Pseudosciaena crocea challenged with Cryptocaryon irritans.

To clarify the effects of a Cryptocaryon irritans infection on the stress, antioxidant and mucosal immune response of the large yellow croaker Pseudosciaena crocea, this study utilized C. irritans at dose of 12,000 (group I); 24,000 (group II); and 36,000 (group III) theronts/fish to infect large yellow croaker weighing 100 ± 10 g. The food intake, survival and relative infection intensity (RII); levels of reactive oxygen species (ROS), malondialdehyde (MDA) and vitamin C (VC), activities of super oxide dismutase (SOD) and catalase (CAT) in liver; variation patterns of lysozyme (LZM), alkaline phosphatase (AKP), complement component 3 (C3) and immunoglobulin M (IgM) levels in the body surface mucus at different time points after infection were compared. These results showed that with the increase of the infection dose and the passage of time, the food intake and survival of the fish gradually decreased. The final survival of the control group (0 theronts/fish), group I, group II, and group III was 100, 100, 96.67 ± 5.77, and 48.33 ± 7.64. Group I, II, and III stopped feeding respectively on the third, third and second days after infection. RII increased significantly with increased infection dose. The RII of the control group, group I, group II, and group III was 0, 0.73 ± 0.06, 1.30 ± 0.26, and 1.84 ± 0.02. With the infection dose increased, ROS contents showed an overall upward trend; MDA contents of the group I, group II and group III did not show significant changes at any timepoint compared with the control group; Activities of SOD and CAT and the overall VC levels in the liver of P. crocea dropped; LZM activity showed an overall upward trend; AKP activity increased first then dropped at each timepoint with its highest level appearing at group II; Complement C3 and IgM levels in body surface mucus were significantly increased. In conclusion, P. crocea has a strong ability to resist oxidative stress caused by the infection of C. irritans. The body surface mucus of P. crocea contains high levels of immune factors, which presented a rapid and significant response to the infection of C. irritans.

Molecular characterization and expression analysis of interferon-gamma in the large yellow croaker Larimichthys crocea.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans.

Effects of Gene Alternative Splicing Events on Resistance to Cryptocaryonosis of Large Yellow Croaker (Larimichthys crocea).

Large yellow croaker (L. crocea) is a productive species in marine aquaculture with great economic value in China. However, the sustainable development of large yellow croaker is hampered by various diseases including cryptocaryonosis caused by Cryptocaryon irritans. The genetic regulation processes for cryptocaryonosis in large yellow croaker are still unclear. In this present study, we analyzed differential alternative splicing events between a C. irritans resistance strain (RS) and a commercial strain (CS). We identified 678 differential alternative splicing (DAS) events from 453 genes in RS and 719 DAS events from 500 genes in CS. A set of genes that are specifically alternatively spliced in RS was identified including mfap5, emp1, and trim33. Further pathway analysis revealed that the specifically alternative spliced genes in RS were involved in innate immune responses through the PRR pathway and the Toll and Imd pathway, suggesting their important roles in the genetic regulation processes for cryptocaryonosis in large yellow croaker. This study would be helpful for the studies of the pathogenesis of cryptocaryonosis and dissection of C. irritans resistance for L. crocea.

Genomic Selection of Large Yellow Croaker (Larimichthys crocea) with a High Plant Protein Diet Enhances the Growth Performance of Offspring.

Fishmeal is over-represented in the diets of large yellow croaker (Larimichthys crocea), and this farming mode, which relies heavily on fishmeal, is highly susceptible to the price of fishmeal and is unsustainable. Therefore, more and more studies on the large yellow croaker tend to replace fishmeal with land-based animal or plant proteins, but few studies have considered it from the genomic selection. In this study, we evaluated the survival rate (SR), final body weight (FBW), body weight gain (BWG), weight gain rate (WGR), and specific growth rate (SGR) of the large yellow croaker GS7 strain, which was obtained through genomic selection for tolerance to plant proteins and analyzed the differences in plant protein utilization between the GS7 strain and unselected commercial large yellow croaker (control group). The results of separate feeding for 60 days showed that although there was no significant difference in SR between the control and GS7 strains (P > 0.05), the BWG, WGR, and SGR of the control were significantly lower (P < 0.05) than those of the GS7 group. Results of mixed feeding after PIT marking showed that compared to the control fish, the GS7 strain had significantly higher BWG, WGR, and SGR (P < 0.0001). To make the experimental results more precise, we compared fishes with equivalent initial body weight (IBW) in the GS7 strain and the control group. The final fish body weight (FBW) of Ctrl-2 (IBW 300-399 g) and Ctrl-4 (IBW 500-599 g) was significantly lower than those of the corresponding GS7-2 and GS7-4 (P < 0.05), while the FBW of Ctrl-1 (IBW 200-299 g) and Ctrl-3 (IBW 400-499 g) was much significantly lower than the corresponding GS7-1 and GS7-3 (P < 0.01). The BWG, WGR, and SGR of Ctrl-1 and Ctrl-4 were more significantly lower than those of the corresponding GS7-1 and GS7-4 (P < 0.01), while the BWG, WGR, and SGR of Ctrl-2 and Ctrl-3 were more significantly different from the corresponding GS7-2 and GS7-3 (P < 0.0001). Our results seem to point toward the same conclusion that the GS7 strain is better adapted to high plant protein diets than the unselected commercial large yellow croaker. These results will provide a reference for the low-fishmeal culture industry of large yellow croakers and the selection and breeding of strains tolerant to a high percentage of plant proteins in other marine fishes.

First Genomic Prediction of Single-Step Models in Large Yellow Croaker.

Genome selection is mainly used in disease-resistant traits of aquatic species; however, its implementation is hindered by a high cost of genotype and phenotype data collection. Single-step genomic best linear unbiased prediction (SSGBLUP) can integrate phenotypes, genetic markers, and pedigree records into simultaneous prediction without increasing genotyping costs. The objective of this study is to investigate the performance of SSGBLUP in large yellow croaker and to evaluate the effects of the number of phenotypic records and genotyping per family on the predictive ability of SSGBLUP. A large yellow croaker population consists of 6898 individuals from 14 families with survival time resistant against Cryptocaryon irritans (C. irritans), body weight (BW), and body length (BL) traits were collected, of which 669 individuals were genotyped. Results showed that the mean predictive ability of all traits in the individuals randomly sampling for SSGBLUP, GBLUP, and BLUP was 0.738, 0.738, and 0.736, respectively. Moreover, the predictive ability of SSGBLUP and BLUP models did not increase with the extra phenotypic records per family, in which the predictive ability of SSGBLUP and BLUP in survival time was 0.853 and 0.851 for only genotyped data (N = 0) used, and 0.852, 0.845 for all phenotypic records (N = 600) used, respectively. However, with the increase in the genotype number of training set, the prediction ability of SSGBLUP and GBLUP model was increased and the highest predictive ability was gained when the genotype number per family was 40 or 45. In addition, the prediction ability of SSGBLUP model was higher than that of GBLUP. Our study showed that the SSGBLUP model still has great potential and advantages in genomic breeding of large yellow croakers. It is recommended that each family provide 100 phenotypic individuals, of which 40 individuals with genotyping data for SSGBLUP model prediction and family resistance evaluation.

Identification and Expression Analysis of LncRNAs Reveal the Immune Mechanism of Visceral White-Nodules Disease Resistance in Large Yellow Croaker.

Long non-coding RNAs (lncRNAs) have several known functions in fish growth processes and signal transduction, but their possible roles in response to bacterial diseases remain largely unresolved. In this study, we report a comprehensive cold-water bacterial disease-responsive lncRNA expression profile for understanding the transcriptional regulatory mechanisms of visceral white-nodules disease resistance in large yellow croaker. A total of 2534 high-confidence lncRNAs were identified by a rigorous filtering pipeline as a basic sequence set for comparative transcriptional analysis. In addition, a total of 10,200 lncRNA-mRNA pairs with high correlation coefficients were identified by expressions level correlation analysis, including non-redundant 381 DE lncRNAs and 2590 differential expressed genes. MSTRG_11084_1 and MSTRG_20402_1 were linked to a large number of target genes and may be involved in important functions in immune regulation. We further revealed the conserved and idiosyncratic features of the disease response process between the technical control strain (TCS) and the resistant strain (RS). Immune-related pathways were enriched in GO terms and KEGG pathways, among which cytokine-cytokine receptor interaction, MAPK signaling pathway, and NF-kappa B signaling pathway may play a key role in VWND resistance in large yellow croaker. Protein-protein interaction network (PPI) analysis revealed that immune-related target genes such as il-10, met, acta2, myc, cav1, and ntrk1, as well as growth and metabolism-related target genes such as pik3r2, igf1, sc5d, hmgcr, and lss were considered the main hub genes. This study represents the first characterization of lncRNAs involved in VWND resistance in large yellow croaker and provides new clues for elucidating the disease response mechanism of large yellow croaker.

Population structure and genome-wide evolutionary signatures reveal putative climate-driven habitat change and local adaptation in the large yellow croaker.

The large yellow croaker (Larimichthys crocea) is one of the most economically valuable marine fish in China and is a notable species in ecological studies owing to a serious collapse of wild germplasm in the past few decades. The stock division and species distribution, which have important implications for ecological protection, germplasm recovery, and fishery resource management, have been debated since the 1960s. However, it is still uncertain even how many stocks exist in this species. To address this, we evaluated the fine-scale genetic structure of large yellow croaker populations distributed along the eastern and southern Chinese coastline based on 7.64 million SNP markers. Compared with the widely accepted stock boundaries proposed in the 1960s, our results revealed that a climate-driven habitat change probably occurred between the Naozhou (Nanhai) Stock and the Ming-Yuedong (Mindong) Stock. The boundary between these two stocks might have shifted northwards from the Pearl River Estuary to the northern area of the Taiwan Strait, accompanied by highly asymmetric introgression. In addition, we found divergent landscapes of natural selection between the stocks inhabiting northern and southern areas. The northern population exhibited highly agminated signatures of strong natural selection in genes related to developmental processes, whereas moderate and interspersed selective signatures were detected in many immune-related genes in the southern populations. These findings establish the stock status and genome-wide evolutionary landscapes of large yellow croaker, providing a basis for conservation, fisheries management and further evolutionary biology studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00165-2.

Genome-Wide Association and Expression Analysis Revealed the Candidate Variants and Molecular Underpinnings of Cold-Stress Response in Large Yellow Croaker.

Large yellow croaker (Larimichthys crocea) is one of the most economically important fish in China. Recently, global climate change has caused more and more intense and extreme low temperature weathers, resulting in huge losses to the large yellow croaker industry. Therefore, it is essential to understand the mechanisms of low-temperature tolerance in large yellow croaker. Here, we conducted an integrative analysis of genome-wide association study (GWAS) and transcriptome analysis to identify candidate variants and reveal the molecular underpinning of cold-stress response in large yellow croaker. A total of 8 significant single nucleotide polymorphisms (SNPs) loci on 6 chromosomes were identified in the GWAS analysis, and 5764 (gill) and 3588 (liver) differentially expressed genes (DEGs) were detected in cold-stressed large yellow croaker, respectively. Further comparative and functional analysis of the candidate genes and DEGs highlighted the importance of pathways/genes related to immune response, cellular stress response, lipid transport, and metabolism in the cold-stress response of large yellow croaker. Our results provide insights into the cold tolerance of large yellow croaker and contribute to genomic-based selection for low-temperature-resistant large yellow croaker.

Disruption of mstn Gene by CRISPR/Cas9 in Large Yellow Croaker (Larimichthys crocea).

The large yellow croaker (Larimichthys crocea) plays an economically vital role in the marine aquaculture in China. Suffering from infection of bacteria and protozoon, effect of extreme weather and stress from high-density farming, genome editing is thought to be an important tool applied to L. croea for enhancing commercial traits such as growth rate, disease resistance, and nutrition component. In this study, we identified two mstn genes in L. croea and investigated the different phylogenetic clades, gene structures, and conserved syntenic relationships. To obtain fast-growing large yellow croaker, we specially selected two validated targets for mstnb knockout, which was homologous to mammalian myostatin gene (MSTN) and downregulated skeletal muscle growth and development. Five significant mutation types were generated in two mosaic mutants by transferring specific CRISPR/Cas9 RNPs (ribonucleoprotein) into the one-cell fertilized embryos based on CRISPR/Cas9 technology. Subsequently, we also elucidated the obstacles and possible measures to improve the success rate of inducing modified large yellow croaker. Our results would provide valuable method and reference for facilitating genome editing programs of the large yellow croaker in the future.