Large Yellow Croaker Research Articles

Lactobacillus plantarum E2 regulates intestinal microbiota and alleviates Pseudomonas plecoglossicida induced inflammation and apoptosis in zebrafish (Danio rerio)

Pseudomonas plecoglossicida infection is a highly contagious epidemic in aquaculture, causing significant mortality among teleost. Our previous research has demonstrated that Lactobacillus plantarum E2 is beneficial for large yellow croaker in resisting infections caused by P. plecoglossicida. However, the relevant mechanisms remain largely unclear. In the present study, we used zebrafish (Danio rerio) to further explore the function of L. plantarum E2 and its mechanisms for resisting P. plecoglossicida infection. E2 supplementation diet significantly improved the growth rates and α-amylase and trypsin activities of the liver in zebrafish. After challenge with P. plecoglossicida strain PQLYC4, the survival rates of zebrafish were improved, and immune-related genes expression (IL-1β, TNF-α, IL-8, Ig-Z, TLR-22 and IL-12α) were down-regulated. Histological analysis showed that E2 group had a longer intestinal villus and thicker intestinal walls after 30 days of feeding and healthier intestinal structure after challenge with P. plecoglossicida strain PQLYC4. Furthermore, co-incubation of zebrafish embryo fibroblast (ZF-4 cells) with L. plantarum E2 reduced apoptosis of ZF-4 cells after exposed to P. plecoglossicida. Intestinal microbiota analysis showed that E2 strain significantly increased the relative abundance of Lactobacillus and Pseudomonas, and PCoA analysis revealed a noticeable divergence in the intestinal microbial communities after E2 supplement. Together, our results suggested that E2 strain may promote zebrafish survival against P. plecoglossicida infection by regulating the intestinal microbiota and alleviating inflammatory response and apoptosis, thus exhibiting the potential as a probiotic.

Effect of gum tragacanth–sodium alginate coatings incorporated with epigallocatechin gallate on the quality and shelf life of large yellow croaker (Larimichthys crocea) during superchilling storage

Abstract The effect of gum tragacanth (GT) and sodium alginate (SA) active coatings incorporated with epigallocatechin gallate (EGCG; 0.16%, 0.32%, and 0.64%, respectively) on the quality of large yellow croaker fillets was evaluated during superchilling storage at –3 ˚C for 42 d. The results revealed that the GT-SA-EGCG active coatings delayed microbial spoilage by reducing the total viable counts of Pseudomonas spp. and psychrophile bacteria and retarded the degradation of the physical properties of water-holding capacity, cooking loss, color, odor and texture. GT-SA-EGCG coatings could also maintain fillet freshness by maintaining lower total volatile basic nitrogen, K value, and histidine content, inhibiting myofibrillar protein degradation during superchilling storage. These results suggested that the active coatings embedded with GT-SA-EGCG could be used as a preservative to enhance the quality of large yellow croaker and prolong the shelf life by 7–14 d during superchilling storage at –3 ˚C.

Genomic selection for hypoxia tolerance in large yellow croaker

The large yellow croaker (Larimichthys crocea) is one of the most economically important marine fishes on the southeast coast of China. However, high-density cultures, inbreeding depression, and environmental pollution have resulted in L. crocea often experiencing hypoxia, which has caused huge economic losses to the L. crocea farming industry. This study aimed to improve the hypoxia tolerance of farmed L. crocea using genomic selection (GS) for breeding. In this study, 399 and 354 samples from the reference and candidate populations, respectively, were genotyped using a 55 K liquid single-nucleotide polymorphism (SNP) array. Based on two different representations (survival status and survival time), the estimated values of heritability for the hypoxia tolerance trait in L. crocea using 38,472 clean SNPs were 0.65 and 0.62, respectively. A comparison of the genomic prediction accuracy under different GS models and SNP densities showed that GBLUP was the best GS model, and the 2500 SNPs selected using a genome-wide association study (GWAS)-based strategy were essentially close to all 38,472 SNPs. Based on the established optimal GS strategy (prediction accuracy was 0.4901 ± 0.0246), genomic estimated breeding values (GEBVs) were calculated for each candidate population. The tolerance capacity tests showed a significant improvement in hypoxia tolerance in the offspring of the candidate population with the top 10% GEBVs, having an 13.40% decrease and a 34.02% increase in the median lethal concentration (LC50) and median lethal time (LT50), respectively, compared to the control group. These results confirmed that GS is an effective genetic breeding technique when selecting for hypoxia tolerance in L. crocea.

Gelation properties and molecular dynamics simulation of thermally induced large yellow croaker (Pseudosciaena crocea) roe protein isolate gels

Roes are the main byproducts during the processing of large yellow croaker (Pseudosciaena crocea) and have not been well utilized. Heat-induced gels were prepared by using P. crocea roe protein isolates (pcRPI), and the influence of pH on the gel properties was also investigated. pcRPI could not form a gel at pH 4–6 but showed gelation behaviors at pH 7–9. The pcRPI gels prepared at pH 8 had a lower gel point (84 °C), and their G′ reached 535.4 Pa. Moreover, pcRPI gels formed at pH 8–9 had a more continuous network structure with a shorter T2 relaxation time than those at other pH conditions. Additionally, vitellogenin and vitellogenin B and C in pcRPI easily formed protein complexes during heating. The hydrophobic interactions, which were the main force between protein molecules during heating, as shown by molecular dynamics simulation, also contributed to gel formation. pcRPI has the potential to be used as a novel hydrogel product in food and other fields.

Molecular characterization of spoilage microbiota in high CO2 refrigerated large yellow croaker (Larimichthys crocea) fillets using metagenomic and metabolomic approaches

Although, the negative role of microorganisms in food safety is well known, the specific role of microorganisms on food is still unclear. The effects of high concentration CO2 on the metabolism of specific spoilage organisms of large yellow croaker and the mechanism of microbial action in the spoilage process of large yellow croaker were investigated. Specific spoilage organisms of large yellow croaker in a refrigerated environment obtained by biodiversity analysis. The effects of spoilage microorganisms on fish fillets were explored using metagenomics and metabolomics approaches. The results showed that the high concentration of carbon dioxide effect prolonged the shelf life of refrigerated large yellow croaker and delayed the deterioration of its quality during storage. Meanwhile, the modified atmosphere packaging with high concentration of carbon dioxide can effectively inhibit the growth of microorganisms, change the structure of spoilage microbial flora, and affect the metabolism of spoilage microorganisms. Pseudomonas spp. and Shewanella spp. had strong spoilage potential and made important contributions to the production of biogenic amines in fish. The contribution of Shewanella spp. to fish spoilage is stronger at high CO2 concentrations. The metabolic capacity of spoilage microorganisms was altered by high CO2 concentration, and the arginine decarboxylase (ADC) pathway was the main pathway for production of putrescine under high CO2 concentration. This study contributes to a better understanding of the role of microorganisms in fish spoilage and provides useful information for the development of new preservation methods.

The design and application of xylose-lysine based time-temperature indicators for visually monitoring the shelf-life of chilled large yellow croaker

The spoilage of seafood is highly dependent on temperature changes during cold chain. To visually reflect the shelf-life of large yellow croakers (0–25 °C), the xylose-lysine based time-temperature indicator (TTI) label was designed and applicated. The ultraviolet (UV) spectrum, and color response of Maillard reaction (MR) indicated that the color changes of TTI was related to the formation of melanoidins of MR. The color changes of TTI, and the quality degradation of large yellow croakers were described by kinetics analysis, which showed that Ea of TVB-N (70.45 kJ/mol) was closer to that of TTI (83.51 kJ/mol). Under fluctuating temperatures, the difference of the effective temperature (Teff) between TVB-N (15.95 °C) and TTI (16.84 °C) was less than 1 °C, and the relative error of shelf-life was −8.33%. Briefly, this novel TTI label as an environmentally friendly system could be attached to sample surface for real-time reflecting shelf-life of seafood in cold chain.

The First High-Quality Chromosome-Level Genome of the Lutjanus erythropterus (Bloch, 1790) Using Single-Tube Long Fragment Reads and Hi-C Technologies.

Lutjanus erythropterus (Bloch, 1790), a Perciformes from the Lutjanidae family, is a commercially important species because of its taste and abundance. Despite the increase in genome resources in recent years, few genome assemblies are available within this fish family for comparative and functional studies. In this study, we determined the chromosomal genome of Crimson snapper using high-throughput Single-Tube Long Fragment Reads sequencing technology and Hi-C data. The final assembly size was 973.04 Mb with contig and scaffold N50 values of 1.51 and 40.65 Mb, respectively. We successfully scaffolded 95.84% of the genome sequence onto 24 chromosomes ranging in length from 19.37 to 49.48 Mb. A total of 22,663 genes and 13,877 gene families were identified in the genome, with 29 gene families being L. erythropterus-specific. A phylogenetic analysis using single-copy gene families showed that L. erythropterus and Larimichthys crocea had the closest genetic relationship with a divergence time of ∼47.7 Ma. This new genomic resource will facilitate comparative genomic studies as well as genetic breeding programs for L. erythropterus.

Characterization and the mechanism underlying the cryoprotective activity of a peptide from large yellow croaker (Pseudosciaena crocea)

Ice crystal-induced protein denaturation is the main cause of the deterioration of fish during frozen storage and transportation. In this study, the ultra-performance liquid chromatography − quadrupole − time of flight (UPLC-Q-TOF) technique was used to identify and screen tryptic peptides Ile-Glu-Glu-Leu-Glu-Glu-Leu-Glu-Ala-Glu-Arg (IEELEEELEAER) from large yellow croaker (Pseudosciaena crocea). The results were used study their cryoprotective effects on turbot fish meat during freeze–thaw cycles at different concentrations, and to investigate their anti-freezing mechanism. The results showed that the I-2.0 group effectively inhibiting the degeneration and structure changes of myofibrillar proteins after three freeze–thaw cycles, and the Ca2+-ATPase activity (1.65 μmolPi/mg/h), increased by 55.86% compared with that of the control group. Additionally, peptide IEELEEELEAER could provide antifreeze protection by binding to the surface of ice crystals and inhibiting their transformation. This peptide acts as a natural cryoprotectant and might be used for the cryogenic storage and transportation of fish products.

Regulation of low-density lipoprotein on lipid metabolism in macrophages of large yellow croaker (Larimichthys crocea)

Low-density lipoprotein (LDL) is the main carrier of cholesterol transport in plasma, which participates in regulating lipid homeostasis. Studies in mammals have shown that high levels of LDL in plasma absorbed by macrophages trigger the formation of lipid-rich foam cells, leading to the development of atherosclerotic plaques. Although lipid-rich atherosclerosis-like lesions have been discovered in the aorta of several fish species, the physiological function of LDL in fish macrophages remains poorly understood. In the present study, LDL was isolated from the plasma of large yellow croaker (Larimichthys crocea), and mass spectrometry analysis identified two truncated forms of apolipoprotein B100 in the LDL protein profile. Transcriptomic analysis of LDL-stimulated macrophages revealed that differentially expressed genes (DEGs) were enriched in various pathways related to lipid metabolism, as confirmed by the fact that LDL increased total cholesterol and cholesteryl esters content. Meanwhile, the gene and protein expression levels of perilipin2 (PLIN2), a DEG enriched in the PPAR signaling pathway, were upregulated in response to LDL stimulation. Importantly, knocking down plin2 significantly attenuates LDL-induced cholesterol accumulation and promotes cholesterol efflux. Furthermore, the transcription factor PPARγ, which is upregulated in response to LDL stimulation, can enhance the promoter activity of plin2. In conclusion, this study suggests that LDL may upregulate plin2 expression through PPARγ, resulting in cholesterol accumulation in fish macrophages. This study will facilitate the investigation of the function of LDL in regulating lipid homeostasis in macrophages and shed light on the evolutionary origin of LDL metabolism in vertebrates.

Mechanism of salt effect on flavor formation in lightly-salted large yellow croaker by integrated multiple intelligent sensory and untargeted lipidomics analyses

Salt has a great influence on food flavor formation. In this study, electronic tongue and nose, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and lipid oxidation levels were used to investigate the influence of different NaCl concentrations on the flavor formation of lightly salted large yellow croaker. The results showed that salt improves the sensory characteristics of the product. Hexanal, 2,5-octanedione, octanal, 1-octen-3-ol, nonanal, and heptanal were key flavor compounds. Phospholipids containing 18-carbon fatty acids are major flavor precursor substances. The TBARS values in samples increase with the increase of salt levels significantly (p < 0.05). Products marinated in 6% NaCl showed the highest lipase activity. Thus, NaCl promotes the hydrolysis and oxidation of phospholipids by increasing lipase activity to produce key flavor substances. This study provides valuable insights into the effects of NaCl on flavor formation, which may help to regulate the flavor of salt-reduced food.

Hydrological connectivity controls on the dynamics of particulate organic matter in a semi-enclosed mariculture bay

Semi-enclosed bay provides favorable conditions for mariculture, however, mariculture activities can disrupt hydrological connectivity of river-coastal continuum and create a bioreactor for particulate organic matter (POM). The impacts of increasing mariculture activities on POM dynamics, including sources, transportation, and transformation, associated dissolved oxygen and nutrients in semi-enclosed bays remain unclear. This study investigated carbon and nitrogen contents, as well as the stable isotopic compositions of POM (δ13CPOC and δ15NPN) in a semi-enclosed bay, Sansha Bay, which is the largest mariculture bay for large yellow croaker in China and is influenced by riverine input and nearshore coastal waters. Our findings revealed that, spatially, riverine POM dominated along main channel that connects river inlet and bay outlet under the influence of water mixing (C:N ratios ∼ 5.7–15.8, δ13CPOC ∼ −21‰--24‰, and δ15NPN ∼ 13‰-17‰). On top of water mixing, autochthonous POM dominates in off-main channel (C:N ratio 5–9, δ13CPOC -21‰--17‰, and δ15NPN 7.0‰-8.5‰). Using a three end-member isotopic mixing model, our data shows that the POM in main channel comprises ∼ 90% of riverine POM and ∼ 10% of phytoplankton production, while in off-main channel, ∼ 59% of POM was contributed by autochthonous phytoplankton production and the rest ∼ 41% was sourced from main channel. Furthermore, riverine POM degradation consumes oxygen along main channel, forming consistent with the classic Redfield stoichiometry of aerobic respiration (N:O2, ∼ 0.13 ± 0.02). In contrast, in off-main channel where water exchange is slower, the degradation of fish feed regenerates dissolved inorganic nitrogen, which subsequently stimulates phytoplankton production, leading to oxygen production following the canonical Redfield stoichiometry of phytoplankton photosynthesis (N:O2, ∼ 0.10 ± 0.01). Our results highlight the influence of hydrological connectivity, superimposed by mariculture activity, in shaping POM biogeochemistry and dissolved oxygen dynamics in semi-enclosed bays. These findings provide important insights for the remediation of risks of hypoxia and harmful algal blooms in coastal mariculture ecosystems.

The heat shock protein 20 gene family in large yellow croaker (Larimichthys crocea): Identification, phylogenetic relationships, expression analyses

Large yellow croaker (Larimichthys crocea) is an economically important fish in China, but its aquaculture industry has been threatened by both biotic and abiotic stressors such as hypoxia and pathogens. In the current study, hsp20 genes were identified and analyzed systematically for the first time from the genome of large yellow croaker, and their roles in hypoxia response and Aeromonas hydrophila, Pseudomonas plecoglossicida infection were investigated. Herein, 11 hsp20 genes were identified and annotated, phylogenetic analysis and selection pressure analysis showed that the hsp20 genes were evolutionarily-constrained and their function was conserved among fishes. Besides, we observed the expression patterns of the hsp20 genes under hypoxia and two pathogens’ stress. In brief, seven, four, seven genes responded to hypoxia stress, A. hydrophila infection and P. plecoglossicida challenge, respectively, which indicated that they were involved in hypoxia and disease responses. Furthermore, pathogen- and time-specific pattern was observed after A. hydrophila and P. plecoglossicida infection whereas tissue-specific pattern was observed after hypoxia exposure, revealing that hsp20 genes showed differential functions in response to hypoxia and immune stress. Taken together, these results provided preliminary information for future analysis of the roles of hsp20 genes in both biotic and abiotic stress response in fish.

Isolation, characterization and virulence of Metanophrys sp. (Ciliophora: Scuticociliatida) from large yellow croaker (Larimichthys crocea) in China

In recent years, an outbreak of large yellow croaker (Larimichthys crocea) disease caused by unknown ciliate infections has occurred along the southeast coast of China, resulting in substantial economic losses. To investigate the taxonomic status and pathogenic characteristics of the ciliate, we isolated the ciliate from the brain tissue of naturally diseased L. crocea larvae, established in vitro monoclonal culture, carried out molecular and morphological studies, and observed the host histopathology. In the present study, we characterized three linked genes (ITS1–5.8S-ITS2, LSU rDNA, SSU rDNA) of the ciliate. The mitochondrial cytochrome oxidase subunit I (mt-COI rDNA) and mitochondrial small subunit ribosome DNA (mt-SSU rDNA) were also applied. The phylogenetic analysis based on ITS1–5.8S-ITS2, LSU, SSU, mt-COI, and mt-SSU rDNA molecular markers showed that the ciliate had the closest genetic relationship with Metanophrys sinensis, and the sequence similarity was 89.95%, 98.22%, 98.74%, 94.63%, and 94.08%, respectively. It was also clustered in a branch with M. sinensis on the phylogenetic tree. Morphological observation showed that the ciliate was melon seed shaped, with a slightly sharp front end and a blunt rear end; a tail cilium, approximately 10 um long; and an oval macronucleus. There were 10–11 somatic kineties, and mitochondria were distributed beneath the cell surface membrane, forming a mitochondrial system that conforms to the morphological characteristics of Metanophrys. After artificially infecting the healthy L. crocea with the ciliate, a large number of ciliates with identical molecular markers and morphology were isolated from the infected L. crocea. The fins and skin of infected fish were partially ulcerated, and the tail or fin base was white, which is the same as the symptoms of the above-mentioned naturally diseased L. crocea. In addition, the results of histopathological observation showed that the ciliate could cause local necrosis and dissolution of L. crocea muscle fibers, infiltration of inflammatory cells in muscle spaces, increased swelling cells in gills and skin, and vacuolation of liver and spleen cells. Therefore, the ciliate that causes the above disease can be classified into the genus Metanophrys, which has been isolated and identified for the first time.

PvgAS: A novel two-component system that controls type III and type VI secretion systems and virulence in the fish pathogen Pseudomonas plecoglossicida

Pseudomonas plecoglossicida is a Gram-negative bacterium that causes chronic granulomatous diseases in fish. However, the molecular mechanisms underlying its pathogenicity are poorly understood. Here, we identified a novel two-component system (TCS) named PvgAS in P. plecoglossicida, which regulates the expression of genes including the type VI secretion system-1 (T6SS-1), a key virulence factor, as well as the type III secretion system (T3SS) gene cluster and several genes associated with metabolic pathways. We showed that PvgAS is essential for bacterial virulence in large yellow croaker (LYC), an important aquaculture species, by controlling T6SS-1 expression, which is crucial for tissue colonization and intracellular survival. In contrast, T3SS was dispensable for P. plecoglossicida virulence in this model. We also confirmed T6SS-1 gene regulation by PvgAS at the transcriptional and translational levels using RNA-seq, RT-qPCR, lacZ reporter, and western blot assays. Our study reveals a regulatory and functional role of PvgAS in P. plecoglossicida pathogenicity and suggests that PvgAS may be a potential target for new therapeutic interventions against this bacterium.

Dietary palm oil enhances Sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis through inducing endoplasmic reticulum stress in muscle of large yellow croaker (Larimichthys crocea).

Sterol regulatory element-binding protein 2 (SREBP2) is considered to be a major regulator to control cholesterol homoeostasis in mammals. However, the role of SREBP2 in teleost remains poorly understand. Here, we explored the molecular characterisation of SREBP2 and identified SREBP2 as a key modulator for 3-hydroxy-3-methylglutaryl-coenzyme A reductase and 7-dehydrocholesterol reductase, which were rate-limiting enzymes of cholesterol biosynthesis. Moreover, dietary palm oil in vivo or palmitic acid (PA) treatment in vitro elevated cholesterol content through triggering SREBP2-mediated cholesterol biosynthesis in large yellow croaker. Furthermore, our results also found that PA-induced activation of SREBP2 was dependent on the stimulating of endoplasmic reticulum stress (ERS) in croaker myocytes and inhibition of ERS by 4-Phenylbutyric acid alleviated PA-induced SREBP2 activation and cholesterol biosynthesis. In summary, our findings reveal a novel insight for understanding the role of SREBP2 in the regulation of cholesterol metabolism in fish and may deepen the link between dietary fatty acid and cholesterol biosynthesis.

The probiotic roles of Lactiplantibacillus plantarum E2 as a dietary supplement in growth promotion and disease resistance of juvenile large yellow croaker (Larimichthys crocea)

Bacterial diseases are the most important limiting factors for the development of large yellow croaker aquaculture. Probiotics are considered to be a promising alternative approach for the control of bacterial diseases in aquaculture. However, the studies on probiotics used in farmed large yellow croakers were very limited. In this study, we isolated and identified a Lactiplantibacillus plantarum E2 from the intestinal tract of large yellow croaker. L. plantarum E2 showed significant antibacterial activities against several aquaculture pathogenic bacteria, intestinal environmental tolerance, and biosafety. After 7 weeks of feeding, the E2 supplementation of dietary significantly improved the growth and the survival rates of large yellow croakers after Pseudomonas plecoglossicida PQLYC4 challenge. Further analysis showed that E2 effectively improved the intestinal integrity, and increased the intestinal α-amylase, trypsin and lipase activities. Moreover, the E2 supplementation also significantly suppressed the mRNA expression of IL-10 and increased the mRNA expression of IL-1β, IL-12α, IL-17D, IFN-γ, and TNF-α-R. Gut microbiota analysis showed that E2 significantly affected gut microbial community composition by decreasing the relative abundance of Sphingomonas and increasing the relative abundance of Lactobacillus and Pseudomonas. Finally, E2 could improve resistance of large yellow croaker against P. plecoglossicida PQLYC4 infection. Therefore, our findings showed that L. plantarum E2 has potential application as a probiotic in large yellow croaker, which may provide a new strategy of preventing and controlling bacterial diseases in this species.

Deep learning to obtain high-throughput morphological phenotypes and its genetic correlation with swimming performance in juvenile large yellow croaker

Breeding for swimming performance in fish may cause changes in morphological traits in offspring, which are not only related to industrial efficiency, but also affect animal welfare. However, the genetic correlation between swimming performance and morphological traits in juvenile large yellow croaker remains unclear, and traditional morphological traits collection is time-consuming and laborious. Deep learning offers new opportunities for high-throughput phenomics, especially for complex morphological traits. In this study, the automatic High-Resolution Network (HRNet) based on a deep learning approach was used for the first time to effectively detect the morphological traits of juvenile large yellow croaker. We assessed the swimming performance (Ucrit) of 1300 juvenile fish, and conducted a genome-wide association study (GWAS) and genetic parameters study on the morphological traits of 383 juvenile fish using a 55 K SNP array. The morphological traits data of 383 fish comes from the predicted results of the HRNet model. The results showed that: (1) All of the 10 morphological traits showed positively correlated with Ucrit, and mostly positive genetic correlated with Ucrit; (2) The heritability of all 10 morphological traits was low to moderate; (3) We identified 4 SNPs significantly associated with morphological traits, and identified candidate genes including sox8, setd6, and ctbp2, and enriched significant pathways including Notch signaling pathway. In conclusion, this study provides an efficient and multi-context adaptive HRNet based on deep learning for high-throughput morphological traits detection of juvenile large yellow croaker, and analyses the genetic basis between morphological traits and swimming performance for this species, which provides a new understanding of the genetic basis of morphological traits of large yellow croaker, and provides a basis for breeding programs.

Non-targeted metabolomics based on liquid chromatography–mass spectrometry to evaluate the metabolites of wild and farmed large yellow croakers (Larimichthys crocea)

Non-targeted metabolomics based on liquid chromatography–mass spectrometry to evaluate the metabolites of wild and farmed large yellow croakers (Larimichthys crocea)

Integration of transcriptomics and metabolomics reveals the effects of sea currents on overwintering of large yellow croaker Larimichthys crocea in cage culture

Large yellow croaker cultured in cages often face overwintering challenges, which have led to significant economic losses in aquaculture. In natural sea areas, apart from low temperatures, sea current is an important factor affecting the overwintering of large yellow croaker. Therefore, we have set up cages with different mesh sizes to investigate the impact of sea currents on the overwintering of large yellow croaker. Three cages with different mesh sizes (0.5 cm, 1.0 cm, and 2.0 cm) were constructed, and 500 fish were placed in each cage. The group with the 0.5-cm mesh served as the control, while the other two groups were experimental (T1 and T2). Water temperature was monitored daily, and water flow rate was measured at the highest tide point of each month (The first and fifteenth days of the lunar calendar). The experiment lasted for 78 days, during which the body weights and lengths of the fish were measured. Muscle samples were taken for body composition analysis, and liver tissues from control and T2 were collected for transcriptome and metabolome analyses. The water temperature ranged from 8.5 to 12.5 °C during the experimental period. The average sea current velocities in the three groups were 0.049 m/s, 0.167 m/s, and 0.286 m/s, respectively. The survival rates of fish in the control, T1 and T2 groups were 85.25%, 80.66%, and 70.32%, respectively. The weight and condition factor decreased significantly in all groups, with the T2 group experiencing the greatest decline. The lipid and protein contents of the fish in the T2 group were significantly lower than those in the control and T1 groups. Transcriptome analysis showed that the insulin and FoxO pathways were related to the regulation of glycolysis/gluconeogenesis. The PPAR signaling pathway was related to the regulation of lipid metabolism. Metabolome analysis revealed 663 significantly different metabolites in the two groups, of which 10 were significantly downregulated and 51 were significantly upregulated. The combined analysis of transcriptome and metabolome showed that both metabolites and genes involved in the ABC transporter pathway were significantly differentially expressed. The results of the study indicated that fish in high flow rate cages require more fat and protein to provide energy. The insulin and PPAR pathways played important roles in regulating glucose and lipid metabolism. The ABC transporter pathway may play important roles in the transport of small molecule substances and participate in energy metabolism during low-temperature activity in large yellow croaker.

Cloning, subcellular localization and antibacterial functional analysis of NK-lysin in yellow drum (Nibea albiflora)

Vibrio harveyi is the primary pathogenic bacteria affecting Nibea albiflora aquaculture. In a previous phase, our laboratory intentionally exposed N. albiflora to V. harveyi and analyzed the outcomes using a combination of genome-wide association study (GWAS) and RNA-seq. The results revealed that the antimicrobial peptide NK-lysin (YdNkl-1) was a candidate gene for resistance to V. harveyi disease in N. albiflora. To investigate the role of the antimicrobial peptide NK-lysin in N. albiflora's antimicrobial immunity, we screened the YdNkl-1 gene from the transcriptome database. The full-length cDNA of YdNkl-1 gene is 508 bp, with an open reading frame (ORF) of 477 bp, encoding 158 amino acids. The deduced amino acid sequence of YdNkl-1 contains a signal peptide (1st-22nd amino acids) and a Saposin B domain (50th-124th amino acids), akin to mammalian NK-lysin. Phylogenetic tree analysis confirmed that the NK-lysin of teleost fish clustered into a single species, and YdNkl-1 was most closely related to Larimichthys crocea. Subcellular localization showed that YdNkl-1 was distributed in cytoplasm and nucleus of yellow drum kidney cells. Furthermore, YdNkl-1 mRNA transcripts were significantly up-regulated in the skin, gill, intestine, head-kidney, liver, and spleen after V. harveyi infection, suggesting a critical role in N. albiflora's defense against V. harveyi infection. Additionally, we purified and observed the YdNkl-1 protein, which exhibited a potent membrane-disrupting effect on V. harveyi, Pseudomonas plecoglossicida, Vibrio parahaemolyticus, Escherichia coli and Bacillus subtilis. These findings underscore the significance of NK-lysin in N. albiflora's resistance to V. harveyi infection and provide new insights into the crucial role of NK-lysin in the innate immunity of teleost fishes.