Mandarin fish von Hippel-Lindau protein regulates the NF-κB signaling pathway via interaction with IκB to promote fish ranavirus replication.

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKß, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.

Hypoxia-inducible factor-1α promotes macrophage functional activities in protecting hypoxia-tolerant large yellow croaker (Larimichthys crocea) against Aeromonas hydrophila infection.

The immune system requires a high energy expenditure to resist pathogen invasion. Macrophages undergo metabolic reprogramming to meet these energy requirements and immunologic activity and polarize to M1-type macrophages. Understanding the metabolic pathway switching in large yellow croaker (Larimichthys crocea) macrophages in response to lipopolysaccharide (LPS) stimulation and whether this switching affects immunity is helpful in explaining the stronger immunity of hypoxia-tolerant L. crocea. In this study, transcript levels of glycolytic pathway genes (Glut1 and Pdk1), mRNA levels or enzyme activities of glycolytic enzymes [hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase A (LDHA)], aerobic respiratory enzymes [pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (IDH), and succinate dehydrogenase (SDH)], metabolites [lactic acid (LA) and adenosine triphosphate (ATP)], levels of bactericidal products [reactive oxygen species (ROS) and nitric oxide (NO)], and transcripts and level changes of inflammatory factors [IL1ß, TNFα, and interferon (IFN) γ] were detected in LPS-stimulated L. crocea head kidney macrophages. We showed that glycolysis was significantly induced, the tricarboxylic acid (TCA) cycle was inhibited, and metabolic reprogramming occurred, showing the Warburg effect when immune cells were activated. To determine the potential regulatory mechanism behind these changes, LcHIF-1α was detected and found to be significantly induced and transferred to the nucleus after LPS stimulation. LcHif-1α interference led to a significant reduction in glycolytic pathway gene transcript expression, enzyme activity, metabolites, bactericidal substances, and inflammatory factor levels; a significant increase in the aerobic respiration enzymes; and decreased migration, invasion, and phagocytosis. Further ultrastructural observation by electron microscopy showed that fewer microspheres contained phagocytes and that more cells were damaged after LcHif-1α interference. LcHif-1α overexpression L. crocea head kidney macrophages showed the opposite trend, and promoter activities of Ldha and Il1ß were significantly enhanced after LcHif-1α overexpression in HEK293T cells. Our data showed that LcHIF-1α acted as a metabolic switch in L. crocea macrophages and was important in polarization. Hypoxia-tolerant L. crocea head kidney showed a stronger Warburg effect and inhibited the TCA cycle, higher metabolites, and bactericidal substance levels. These results collectively revealed that LcHif-1α may promote the functional activities of head kidney macrophages in protecting hypoxia-tolerant L. crocea from Aeromonas hydrophila infection.

Genome-wide characterization, phylogenetic and expression analysis of Galectin gene family in Golden pompano Trachinotus ovatus.

Galectins (Gals) are a type of S-type lectin that are widespread and evolutionarily conserved among metazoans, and can act as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). In this study, 10 Gals (ToGals) were identified in the Golden pompano (Trachinotus ovatus), and their conserved domains, motifs, and collinearity relationships were analyzed. The expression of ToGals was regulated following infection to Cryptocaryon irritans and Streptococcus agalactiae, indicating that ToGals participate in immune responses against microbial pathogens. Further analysis was conducted on one important member, Galectin-3, subcellular localization showing that ToGal-3like protein is expressed both in the nucleus and cytoplasm. Recombinant protein obtained through prokaryotic expression showed that rToGal-3like can agglutinate red blood cells of rabbit, carp and golden pompano and also agglutinate and kill Staphylococcus aureus, Bacillus subtilis, Vibrio vulnificus, S. agalactiae, Pseudomonas aeruginosa, and Aeromonas hydrophila. This study lays the foundation for further research on the immune roles of Gals in teleosts.

Daily crowding stress has limited, yet detectable effects on skin and head kidney gene expression in surgically tagged atlantic salmon (Salmo salar).

To ensure welfare-friendly and effective internal tagging, the tagging process should not cause a long-term burden on individuals given that tagged fish serve as representatives for the entire population in telemetry applications. To some extent, stress is inevitable within regular aquaculture practices, and thus, the consequences of long-term stress should be described in terms of their effects on internal tagging. In fish, stressors activate the Hypothalamus-Pituitary-Interrenal (HPI) and Brain-Sympathetic-Chromaffin Cell (BSC) axes, leading to neuroimmunoendocrine communication and paracrine interactions among stress hormones. The interrelation between wound healing and stress is complex, owing to their shared components, pathways, and energy demands. This study assessed 14 genes (mmp9, mmp13, il-2, il-4, il-8a, il-10, il-12, il-17d, il-1b, tnfa, ifng, leg-3, igm, and crh) in the skin (1.5 cm from the wound) and head kidney over eight weeks. These genes, associated with cell signaling in immunity, wound healing, and stress, have previously been identified as influenced and regulated by these processes. Half of a group of Atlantic salmon (n = 90) with surgically implanted dummy smart-tags were exposed to daily crowding stress. The goal was to investigate how this gene panel responds to a wound alone and then to the combined effects of wounding and daily crowding stress. Our observations indicate that chronic stress impacts inflammation and impedes wound healing, as seen through the expression of matrix metalloproteinases genes in the skin but not in the head kidney. This difference is likely due to the ongoing internal wound repair, in contrast to the externally healed wound incision. Cytokine expression, when significant in the skin, was mainly downregulated in both treatments compared to control values, particularly in the study's first half. Conversely, the head kidney showed initial cytokine downregulation followed by upregulation. Across all weeks observed and combining both tissues, the significantly expressed gene differences were 12 % between the Wound and Stress+ groups, 28 % between Wound and Control, and 25 % between Stress+ and Control. Despite significant fluctuations in cytokines, sustained variations across multiple weeks are only evident in a few select genes. Furthermore, Stress+ individuals demonstrated the most cytokine correlations within the head kidney, which may suggest that chronic stress affects cytokine expression. This investigation unveils that the presence of stress and prolonged activation of the HPI axis in an eight weeklong study has limited yet detectable effects on the selected gene expression within immunity, wound healing, and stress, with notable tissue-specific differences.

Multiple effects of dietary supplementation with Lactobacillus reuteri and Bacillus subtilis on the growth, immunity, and metabolism of largemouth bass (Micropterus salmoides).

Probiotics play an essential role in the largemouth bass (Micropterus salmoides) aquaculture sector. They aid the fish in sickness prevention, intestinal structure improvement, food absorption, and immune system strengthening. In this experiment, Bacillus subtilis (BS, 107 CFU/g) and Lactobacillus reuteri (LR, 107 CFU/g) were added to the feed and then fed to M. salmoides for 35 days. The effects of two probiotics on the growth, immunity, and metabolism of M. salmoides organisms were studied. The results revealed that the BS group significantly increased the growth rate and specific growth rate of M. salmoides, while both the BS and LR groups significantly increase the length of villi M. salmoides intestines. The BS group significantly increased the levels of AKP, T-AOC, and CAT in the blood of M. salmoides, as well as AKP levels in the intestine. Furthermore, the BS group significantly increased the expression of intestinal genes Nrf2, SOD1, GPX, and CAT, while significantly decreasing the expression of the keap1 gene. M. salmoides gut microbial analysis showed that the abundance of Planctomycetota was significantly different in both control and experimental groups. Analyzed at the genus level, the abundance of Citrobacter, Paracoccus, Luedemannella， Sphingomonas, Streptomyces and Xanthomonas in the both control and experimental groups were significantly different. The BS group's differentially expressed genes were predominantly enriched in oxidative phosphorylation pathways in the intestine, indicating that they had a good influence on intestinal metabolism and inflammation suppression. In contrast, differentially expressed genes in the LR group were primarily enriched in the insulin signaling and linoleic acid metabolism pathways, indicating improved intestine metabolic performance. In conclusion, B. subtilis and L. reuteri improve the growth and health of M. salmoides, indicating tremendous potential for enhancing intestinal metabolism and providing significant application value.

Molecular characterization of the SP3a gene, a negative regulator of viral infection in the orange-spotted grouper, Epinephelus coioides.

SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry.

Gut Microbiome-Driven metabolites influence skin pigmentation in TYRP1 mutant Oujiang Color Common Carp.

The gut microbiome plays a key role in regulating the gut-skin axis, and host genetics partially influence this regulation. The study investigated the role of gut microbiota and host genetics in the gut-skin axis, focusing on the unusual "coffee-like" color phenotype observed in TYRP1 mutant Oujiang Color Common Carp. We employed comparative high-throughput omics data from wild-type and mutant fish to quantify the influence of both genetics and gut microbes on skin transcriptomic expression and blood metabolites. We found 525 differential metabolites (DMs) and 45 distinct gut microbial genera in TYRP1 mutant fish compared to wild type. Interaction and causal mediation analyses revealed a complex interplay. The TYRP1 mutation likely triggers an inflammatory pathway involving Acinetobacter bacteria, Leukotrience-C4 and Spermine. This inflammatory response appears to be counterbalanced by an anti-inflammatory cardiovascular genetic network. The net effect is the upregulation of COMT, PLG, C2, C3, F10, TDO2, MHC1, and SERPINF2, leading to unusual coffee-like coloration. This study highlights the intricate interplay between gut microbiota, host genetics, and metabolic pathways in shaping complex phenotypes.

A conserved phenylalanine motif among teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG-EPG(B.g.)-the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Analyses of the Mx family members in lumpfish: Molecular characterization, phylogeny, and gene expression analyses.

Members of the myxovirus resistance (Mx) protein family play an essential role in antiviral immunity. They are Dynamin-like GTPases, induced by interferons. In the current study, we have characterized two predicted MX genes (MX1 and MX2) from lumpfish (Cyclopterus lumpus L.), having 12 and 13 exons, respectively. Mx2 has two isoforms (Mx2-X1 and Mx2-X2) which differ in exon 1. The lumpfish Mx proteins contain an N-terminal Dynamin-like GTPase domain, the middle domain (MD) and GTPase effector domain (GED) characteristic for Mx proteins. Phylogenetic analyses grouped all the lumpfish Mx sequences in group 1, and synteny analyses showed that both genes were localized at chromosome 5 in proximity to the genes Tohc7, Atxn7 and Psmd6. In vitro stimulation experiment showed that both MX1 and MX2-X2 were highly upregulated upon exposure to poly(I:C), but not bacteria, 24 h post exposure, indicating their role in antiviral immunity.

A leukocyte immune-type receptor specific polyclonal antibody recognizes goldfish kidney leukocytes and activates the MAPK pathway in isolated goldfish kidney neutrophil-like cells.

Leukocyte immune-type receptors (LITRs) belong to a large family of teleost immunoregulatory receptors that share phylogenetic and syntenic relationships with mammalian Fc receptor-like molecules (FCRLs). Recently, several putative stimulatory Carassius auratus (Ca)-LITR transcripts, including CaLITR3, have been identified in goldfish. CaLITR3 has four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain containing a positively charged histidine residue, and a short cytoplasmic tail region. Additionally, the calitr3 transcript is highly expressed by goldfish primary kidney neutrophils (PKNs) and macrophages (PKMs). To further investigate the immunoregulatory potential of CaLITR3 in goldfish myeloid cells, we developed and characterized a CaLITR3-epitope-specific polyclonal antibody (anti-CaL3.D1 pAb). We show that the anti-CaL3.D1 pAb stains various hematopoietic cell types within the goldfish kidney, as well as in PKNs and PKMs. Moreover, cross-linking of the anti-CaL3.D1-pAb on PKN membranes induces phosphorylation of p38 and ERK1/2, critical components of the MAPK pathway involved in controlling a wide variety of innate immune effector responses such as NETosis, respiratory burst, and cytokine release. These findings support the stimulatory potential of CaLITR3 proteins as activators of fish granulocytes and pave the way for a more in-depth examination of the immunoregulatory functions of CaLITRs in goldfish myeloid cells.

Innate and adaptive immune response of Rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri.

Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1ß and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study.

Preliminary study of BF/C2 on immune mechanism of grass carp against GCRV infection.

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.

Role of the Pseudomonas plecoglossicida fliL gene in immune response of infected hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

Pseudomonas plecoglossicida, a gram-negative bacterium, is the main pathogen of visceral white-point disease in marine fish, responsible for substantial economic losses in the aquaculture industry. The FliL protein, involved in torque production of the bacterial flagella motor, is essential for the pathogenicity of a variety of bacteria. In the current study, the fliL gene deletion strain (ΔfliL), fliL gene complement strain (C-ΔfliL), and wild-type strain (NZBD9) were compared to explore the influence of the fliL gene on P. plecoglossicida pathogenicity and its role in host immune response. Results showed that fliL gene deletion increased the survival rate (50%) and reduced white spot disease progression in the hybrid groupers. Moreover, compared to the NZBD9 strain, the ΔfliL strain was consistently associated with lower bacterial loads in the grouper spleen, head kidney, liver, and intestine, coupled with reduced tissue damage. Transcriptomic analysis identified 2 238 differentially expressed genes (DEGs) in the spleens of fish infected with the ΔfliL strain compared to the NZBD9 strain. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the DEGs were significantly enriched in seven immune system-associated pathways and three signaling molecule and interaction pathways. Upon infection with the ΔfliL strain, the toll-like receptor (TLR) signaling pathway was activated in the hybrid groupers, leading to the activation of transcription factors (NF-κB and AP1) and cytokines. The expression levels of proinflammatory cytokine-related genes IL-1ß, IL-12B, and IL-6 and chemokine-related genes CXCL9, CXCL10, and CCL4 were significantly up-regulated. In conclusion, the fliL gene markedly influenced the pathogenicity of P. plecoglossicida infection in the hybrid groupers. Notably, deletion of fliL gene in P. plecoglossicida induced a robust immune response in the groupers, promoting defense against and elimination of pathogens via an inflammatory response involving multiple cytokines.

Genome-Wide Identification and Interaction Analysis of Turbot Heat Shock Protein 40 and 70 Families Suggest the Mechanism of Chaperone Proteins Involved in Immune Response after Bacterial Infection.

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

ROS-Induced Autophagy of Skeletal Muscle Confers Resistance of Rice Flower Carp (Cyprinus carpio) to Short-Term Fasting.

Starvation is one of the main stresses for fish due to food shortage, the evasion of predators, and intraspecific competition. This research evaluated the impact of brief fasting periods on reactive oxygen species (ROS) levels, antioxidant response, mRNA expression of antioxidants, autophagy-related signaling genes, and autophagosome development in the muscle tissue of rice flower carp. Following a three-day fasting period, the levels of ROS and MDA rose. Additionally, after 3 d of fasting, there was a notable upregulation of NRF2 and significant increases in the levels of GSH and the activities of enzymes such as SOD, CAT, GST, GR, and GPX, while the expression of the autophagy marker gene LC3B did not change (p < 0.05). After 7 d of fasting, the content of the ROS, the activity of SOD and GR, and the GSH content reached the maximum (p < 0.05). Concurrently, there was a significant rise in the quantity of autophagosomes. An RT-qPCR analysis revealed that seven d of starvation significantly elevated the mRNA expression of genes associated with the initiation and expansion of autophagosome membranes, vesicle recycling, and cargo recruitment, including ULK1, BECLIN1, LC3B, ATG3, ATG4B, ATG4C, ATG5, ATG9, and P62. After feeding resumed for 3 d, the mRNA level of BECLIN1, ATG3, ATG4B, ATG4C, ATG5, LC3B, and P62 still remained at a high level. The LC3II protein reached its highest level. All autophagy-related gene expression decreased in the 7-day resumed feeding group. Our data implied that short-term fasting can cause oxidative stress and disrupt the antioxidant system first and then induce autophagy in the muscles of rice flower carp. These findings shed light on how fasting affects muscle homeostasis in fish. ROS-induced autophagy of the skeletal muscle may confer the resistance of rice flower carp to short-term fasting.

Comparative Transcriptome Analysis of the Hypothalamic-Pituitary-Gonadal Axis of Jinhu Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂) and Tiger Grouper (Epinephelus fuscoguttatus).

Jinhu groupers, the hybrid offspring of tiger groupers (Epinephelus fuscoguttatus) and potato groupers (Epinephelus tukula), have excellent heterosis in fast growth and strong stress resistance. However, compared with the maternal tiger grouper, Jinhu groupers show delayed gonadal development. To explore the interspecific difference in gonadal development, we compared the transcriptomes of brain, pituitary, and gonadal tissues between Jinhu groupers and tiger groupers at 24-months old. In total, 3034 differentially expressed genes (DEGs) were obtained. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that the osteoclast differentiation, oocyte meiosis, and ovarian steroidogenesis may be involved in the difference in gonadal development. Trend analysis showed that the DEGs were mainly related to signal transduction and cell growth and death. Additionally, differences in expression levels of nr4a1, pgr, dmrta2, tbx19, and cyp19a1 may be related to gonadal retardation in Jinhu groupers. A weighted gene co-expression network analysis revealed three modules (i.e., saddlebrown, paleturquoise, and greenyellow) that were significantly related to gonadal development in the brain, pituitary, and gonadal tissues, respectively, of Jinhu groupers and tiger groupers. Network diagrams of the target modules were constructed and the respective hub genes were determined (i.e., cdh6, col18a1, and hat1). This study provides additional insight into the molecular mechanism underlying ovarian stunting in grouper hybrids.

Hepatopancreas Transcriptome Analysis of Spinibarbus sinensis to Reveal Different Growth-Related Genes.

Spinibarbus sinensis, also known as Qingbo, is an important economic fish in China. However, the detailed mechanisms underlying its growth are still unknown. To excavate the genes and signaling pathways related to its growth, we compared the transcriptome profiles of the hepatopancreas tissues of S. sinensis, with two groups of growth rate for evaluation. An average of 66,304,909 and 68,739,585 clean reads were obtained in the fast growth (FG) and slow growth (SG) group, respectively. The differential gene expression analysis results showed that 272 differentially expressed genes (DEGs) were screened between the FG and SG groups, including 101 up-regulated genes and 171 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results showed that GO terms related to metabolic process, organic substance metabolic process, and catalytic activity were enriched, pathway signals related to steroid biosynthesis and protein digestion and absorption were also detected. Meanwhile, the potential key regulatory genes sst2, fndc4, and cckra related to the growth of S. sinensis were screened. Reverse transcript fluorescence quantitative PCR (RT-qPCR) validation of 18 DEGs associated with growth differences showed that the RT-qPCR results were consistent with RNA-seq analysis, and nine genes, stk31, gpr149, angptl1, fstl1, sik1, ror2, nlrc3, pdlim2, and nav2 were significantly expressed in the FG group. bmp1, stc1, gpatch8, sstrt2, s100a1, ktf6, cckar6, sync1, bhlha15, a total of nine genes were significantly expressed in the SG group. This study provides basic information for improving the growth characteristics of S. sinensis and the functional research of candidate genes.

Sws2 Gene Positively Regulates Melanin Production in Plectropomus leopardus Skin via Direct Regulation of the Synthesis of Retinoic Acid.

Opsins are a class of transmembrane proteins encoded by opsin genes, and they play a variety of functional roles. Short wavelength-sensitive opsin 2 (sws2), one of the five classes of visual opsin genes, mainly senses blue light. Previous research has indicated that sws2 is essential for melanocyte formation in fish; however, its specific role in skin color differentiation remains to be elucidated. Here, we identified the sws2 gene in a prized reef-dwelling fish, Plectropomus leopardus. The full-length P. leopardus sws2 gene encodes a protein consisting of 351 amino acids, and exhibits substantial homology with other fish species. The expression of the sws2 gene was widespread across P. leopardus tissues, with high expression in eye and skin tissues. Through immunohistochemistry and in situ hybridization analyses, we discovered that the sws2 gene was primarily localized in the rod and cone cells of the retina, and epidermal cells of the skin. Furthermore, dsRNA interference was used for sws2 gene knockdown in living P. leopardus to elucidate its function in skin color differentiation. Black-color-related genes, melanin contents, and tyrosinase activity in the skin significantly decreased after sws2 knockdown (p < 0.05), but red-color-related genes and carotenoid and lutein contents significantly increased (p < 0.05). Retinoic acid injection produced the opposite results. Our results suggested that the sws2 gene influences P. leopardus skin color regulation by affecting vitamin synthesis and melanin-related gene expression levels. This study establishes a foundation for elucidating the molecular mechanisms by which sws2 regulates melanocyte formation in fish skin.

Transcriptomic and Epigenomic Responses to Cortisol-Mediated Stress in Rainbow Trout (Oncorhynchus mykiss) Skeletal Muscle.

The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.

CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.