Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Efficient RNA Virus Targeting via CRISPR/CasRx in Fish.

The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.

Effects of nanoplastic exposure on the immunity and metabolism of red crayfish (Cherax quadricarinatus) based on high-throughput sequencing.

Previous studies have shown that nanoplastics (NPs) are harmful pollutants that threaten aquatic organisms and ecosystems, however, less research has been conducted on the hazards of NPs for aquaculture animals. In this study, Cherax quadricarinatus was used as an experimental model to evaluate the possible effects of three concentrations (25, 250 and 2500 µg/L) of NPs on red crayfish. The toxicological effects of NPs on this species were investigated based on transcriptomics and microbiome. A total of 67,668 genes were obtained from the transcriptome. The annotation rate of the four major libraries (Nr, KEGG, KOG, Swissprot) was 40.17 %, and the functions of differential genes were mainly related to antioxidant activity, metabolism and immune processes. During the experiment, the activities of superoxide dismutase (SOD) and catalase (CAT) in the high concentration group were significantly decreased, while the concentration of malondialdehyde (MDA) increased after nanoplastics (NPs) exposure, and SOD1, Jafrac1 were significantly reduced at high concentrations. expression is inhibited. The immune genes LYZ and PPO2 were highly expressed at low concentrations and suppressed at high concentrations. After 14 days of exposure to NPs, significant changes in gut microbiota were observed, such as decreased abundances of Actinobacteria, Bacteroidetes, and Firmicutes. NPs compromise host health by inducing changes in microbial communities and the production of beneficial bacterial metabolites. Overall, these results suggest that NPs affect immune-related gene expression and antioxidant enzyme activity in red crayfish and cause redox imbalance in the body, altering the composition and diversity of the gut microbiota.

Cloning and characterization of rec8 gene in orange-spotted grouper (Epinephelus coioides) and Dmrt1 regulation of rec8 promoter activity.

Meiosis is a specialized type of cell division critical for gamete production during sexual reproduction in eukaryotes. The meiotic recombination protein Rec8 has been identified as an important factor in germ cell meiotic initiation in vertebrates; however, its equivalent role in teleosts is poorly characterized. In this study, we cloned and sequenced the rec8 gene from orange-spotted grouper (Epinephelus coioides). The cDNA sequence consisted of 2244 base pairs (bp), including a 5' untranslated region (UTR) of 198 bp and a 3'UTR of 284 bp. The open reading frame of grouper rec8 was 1752 bp, encoding 584 amino acids. Expression levels of rec8 were higher in the ovary, intersex gonad, and testis. A neighbor-joining phylogenetic tree based on the deduced amino acid sequence indicated a common origin for grouper and other teleost rec8 molecules. Immunohistochemistry using a polyclonal anti-Rec8 antibody localized the protein in the oogonia and primary oocytes in the ovary and in spermatogonia and spermatocytes in the intersex gonad and testis, suggesting that Rec8 may play an important role in the meiotic division and the development of grouper germ cells. In addition, we found that the transcription factor Dmrt1 increased rec8 promoter activity through the second binding site, based on dual-luciferase assays. Together, these results suggest that Rec8 plays a crucial role in meiosis and may be regulated by Dmrt1 to affect meiosis in groupers.

Polystyrene nanoplastics affect digestive function and growth in juvenile groupers.

Polystyrene nanoplastics (PS-NPs) can impair antioxidant, immune, and nervous system functions as well as growth and development in aquatic organisms. At present, however, little is known about the effects and underlying mechanisms of PS-NPs on the digestive system of marine fish. Here, we studied the effects of these plastics on the intestinal health and growth performance of juvenile orange-spotted groupers (Epinephelus coioides). Based on histopathological analysis, we found that the liver and intestines can uptake PS-NPs at exposure concentrations of 300 and 3000 µg/ml, respectively. After 14 d of exposure, the activities of digestive enzymes lipase (LPS), trypsin (TRS), and lysozyme (LZM) were reduced, indicating that PS-NPs negatively affected digestive function in juvenile groupers. The PS-NPs also altered microbial community composition, resulting in a decrease in diversity and simplification of network relationships in the intestinal microbiota, but a significant increase in certain harmful bacteria, especially Vibrio and Aliivibrio. In addition, community assembly changed from being driven primarily by deterministic processes (68.89% for control group) to stochastic processes (73.33% and 51.11% for 300 and 3000 µg/ml PS-NP exposure groups, respectively). Furthermore, the specific growth rate (SGR) of the juvenile orange-spotted groupers decreased significantly with increasing PS-NP exposure concentrations (0.158% ± 0.032%, 0.095% ± 0.020%, and 0.074% ± 0.016% for 0, 300, and 3000 µg/L PS-NP groups, respectively). These results suggest that marine PS-NPs are harmful to the digestive system of juvenile fish and highlight the importance of evaluating the long-term impact of NPs in reshaping marine populations.

Liposome-Encapsulated Rec8 and Dmrt1 Plasmids Induce Red-Spotted Grouper (Epinephelus akaara) Testis Maturation.

In fish, the maturity of gonads plays an important role in the development and reproduction of the population, and it also dictates the success of captive breeding. Therefore, finding ways to promote gonadal maturation is an important goal in aquaculture. In this study, we injected recombinant dmrt1 and rec8 overexpression plasmids packaged in liposomes into the immature testis of red-spotted grouper (Epinephelus akaara) and measured the expression of Dmrt1 and Rec8 protein in vivo. Gonadosomatic index (GSI) and gonadal histology analyses showed that the testis developed from the immature to the mature state within 7 days after plasmid injection. Additionally, the spermatozoa concentration and motility in plasmid-injected fish was the same as that of naturally mature fish. These results provided evidence that delivery of dmrt1 and rec8 expression plasmids into the testis via injection induced testis maturation in vivo.

The ERß-CXCL19/CXCR4-NFκB pathway is critical in mediating the E2-induced inflammation response in the orange-spotted grouper (Epinephelus coioides).

The main physiological function of 17ß-estradiol (E2) in vertebrates is to regulate sexual development and reproduction. In fish, especially hermaphroditic fish, estrogen is often used to aid reproduction, but it also can trigger an inflammatory response. However, the molecular mechanism for this E2-induced inflammatory reaction is not clear. In this study, we found that the ERß-CXCL19/CXCR4-NFκB cascade regulated the E2-induced inflammatory response in the orange-spotted grouper (Epinephelus coioides). Strikingly, E2 treatment resulted in significantly high expression of inflammatory cytokines and induced phosphorylation and degradation of IκBα and translocation of NFκB subunit p65 to the nucleus in grouper spleen cells. However, the E2-induced inflammatory response could be prevented by the broad estrogen receptor (ER) ligand ICI 182,780. Moreover, the luciferase assay showed that E2 induced the inflammatory response by activating the promotor of chemokine CXCL19 through ERß1 and ERß2. Knockdown of CXCL19 blocked the E2-induced inflammatory response and NFκB nucleus translocation. Additionally, knockdown of chemokines CXCR4a and CXCR4b together, but not alone, blocked the E2-induced inflammatory response. The immunofluorescence assay and co-immunoprecipitation analysis showed that CXCL19 mediated the E2-induced inflammatory response by activating CXCR4a or CXCR4b. Taken together, these results showed that the ERß-CXCL19/CXCR4-NFκB pathway mediated the E2-induced inflammatory response in grouper. These findings are valuable for future comparative immunological studies and provide a theoretical basis for mitigating the adverse reactions that occur when using E2 to help fish reproduce.

Polystyrene nanoplastics alter virus replication in orange-spotted grouper (Epinephelus coioides) spleen and brain tissues and spleen cells.

Polystyrene nanoplastics (PS-NPs) are known to impair the function of the digestive system, intestinal flora, immune system, and nervous system of marine organisms. We tested whether PS-NPs influence viral infection of orange-spotted grouper (Epinephelus coioides). We found that grouper spleen (GS) cells took up PS-NPs at exposure concentrations of 5, 50, and 500 µg/mL and experienced cytotoxicity at 50 and 500 µg/mL concentrations. At 12 h after exposure to 50 µg/mL of PS-NPs, the replication of Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) increased in GS cells after their invasion. Juvenile fish exposed to 300 and 3000 µg/L of PS-NPs for 7 d showed PS-NPs uptake to the spleen and vacuole formation in brain tissue. Moreover, PS-NPs exposure accelerated SGIV replication in the spleen and RGNNV replication in the brain. PS-NP exposure also decreased the expression of toll-like receptor genes and interferon-related genes before and after virus invasion in vitro and in vivo, thus reducing the resistance of cells and tissues to viral replication. This is the first report that PS-NPs have toxic effects on GS cells and spleen and brain tissues, and it provides new insights into assessing the impact of PS-NPs on marine fish.

l-arginine inhibited apoptosis of fish leukocytes via regulation of NF-κB-mediated inflammation, NO synthesis, and anti-oxidant capacity.

The increased apoptosis plays an important role in bacterial invasion. In addition, LPS can induce inflammation and apoptosis of leukocytes via the production of reactive oxygen and nitrogen species. In the present study, we investigated the potential protective role of l-arginine (L-Arg) against the apoptosis of fish leukocytes in vitro. The results of Annexin V-FITC/PI staining and TUNEL assay indicated that L-Arg significantly alleviated the apoptosis of fish leukocytes induced by LPS at 24 h and 72 h post incubation (hpi). High caspase-3 activities induced by LPS at 72 hpi were significantly inhibited by L-Arg. Moreover, L-Arg supplementation also significantly decreased the mRNA expression levels of caspases at most time points, which contributed to the anti-apoptotic roles of L-Arg. Further analysis showed that L-Arg significantly inhibited the expression of several pro-inflammatory cytokines including IL-8 and TNF-α, partially via the down-regulation of the genes involved in NF-κB/MyD88 including NF-κB, IKKα and IKKγ. The down-regulation of these pro-inflammatory cytokines by L-Arg supplementation led to the further decrease in the expression of death receptor FasL, contributing to the anti-apoptotic effect of L-Arg. In addition, L-Arg supplementation increased both iNOS mRNA expression and NO production. The mRNA expressions of several anti-oxidant enzymes including SOD, CAT and GSHPx were also significantly increased after L-Arg supplementation, which accelerated the clearance of reactive oxygen species. In all, L-Arg inhibited apoptosis of fish leukocytes both via the increased NO production and antioxidant capacity and via the inhibition of inflammation mediated by NF-κB/MyD88 pathway.