A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1β, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases.

A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder (Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events (12,352) than the R group (11,452). The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.

Freshwater scarcity makes saline–alkaline water essential for sustainable aquaculture. Current research has primarily focused on individual salinity or alkalinity effects on fish, with limited studies addressing their interactive effects. We found significant synergistic toxicity between salinity and alkalinity (r ≈ −0.950/−0.925) in largemouth bass (Micropterus salmoides), demonstrating higher salinity levels corresponding to lower 96-h median lethal concentration (96 h LC₅₀) values for alkalinity, and vice versa. A subsequent 56-day subacute stress trial (salinity: 6‰; alkalinity: 20 mmol/L) assessed the impact on largemouth bass through growth efficiency, histopathology, biochemical assays, transcriptomics, and metabolomics, comparing a saline–alkaline group (SA) with a normal control group (NC). There were no significant differences in growth or survival between the SA and NC groups, but the SA group exhibited pathological changes in gill and liver tissues. Biochemically, the SA group exhibited elevated malondialdehyde, glutathione, and blood urea nitrogen levels, whereas glutathione peroxidase activity significantly decreased. Integrated transcriptomics and metabolomics analyses demonstrated that saline–alkaline stress disrupts lipid, amino acid, and steroid metabolism in largemouth bass, affecting steroid biosynthesis, fatty acid metabolism, glycerophospholipid metabolism, and cysteine and methionine metabolism pathways. Fish adapt by adjusting gene expression and metabolite levels to maintain metabolic balance. This study highlights adaptive mechanisms and applications for sustainable largemouth bass culture in saline–alkaline environments.

The roughskin sculpin (Trachidermus fasciatus) is an endangered fish species in China. In recent years, artificial breeding technology has made significant progress, and the population of roughskin sculpin has recovered in the natural environment through enhancement programs and the release of juveniles. However, the effects of released roughskin sculpin on the genetic structure and diversity of wild populations remain unclear. Studies on genetic diversity analysis based on different types and numbers of molecular markers have yielded inconsistent results. In this study, we obtained 2,610,157 high-quality SNPs and 494,698 InDels through whole-genome resequencing of two farmed populations and one wild population. Both farmed populations showed consistent levels of genomic polymorphism and a slight increase in linkage compared with wild populations. The population structure of the two farmed populations was distinct from that of the wild population, but the degree of genetic differentiation was low (overall average Fst = 0.015). Selective sweep analysis showed that 523,529 genes were selected in the two farmed populations, and KEGG enrichment analysis showed that the selected genes were related to amino acid metabolism, which might be caused by artificial feeding. The findings of this study provide valuable additions to the existing genomic resources to help conserve roughskin sculpin populations.

Since the advent of germ cell transplantation (GCT), it has been widely used in shortening the fish breeding cycle, sex-controlled breeding and the protection of rare and endangered fish. In this study, the effectiveness of female sterile recipient preparation and donor stem cell isolation and purification were comprehensively evaluated for spermatogonial stem cell transplantation (SSCT) in Paralichthys olivaceus. The best way to prepare sterile recipients was found to be giving three-year-old fish four intraovarian injections of busulfan (20 mg/kg body weight) combined with exposure to a high temperature (28 °C) after the spawning season compared with the two other ways, which induced apoptosis of most of the endogenous germ cells, resulting in shrinkage of the spawning plate and enlargement of the ovarian lumen. Further analysis showed that both the gonadosomatic index and germ-cell-specific vasa expression were significantly lower than those of the natural-temperature group before treatment (p < 0.05). A high percentage (>60.00%) of spermatogonial stem cells (SSCs) were obtained after isolation and purification and were transplanted into the prepared recipients. After three weeks of SSCT, the numbers of PKH26-labeled SSCs were increased in the ovaries of the recipients. These findings provide a basis for the establishment of an ideal SSCT technique using P. olivaceus females as the recipients, ultimately contributing to the efficient conservation of male germplasm resources and effective breeding.

The cryopreservation and transplantation of germline stem cells (GSCs) have become the key to conserving fish genetic resources and safeguarding species diversity. This study aimed to investigate the effects of post-mortem temperature and time on the preservation of oogonial stem cells (OSCs) in the marine fish Paralichthys olivaceus. OSCs remained viable after fish death, and they remained viable and could be cultured after storage at 19 °C for 15 h and at 4 °C for 24 h. Combined transcriptomic and metabolomic analysis was used to identify the pathways leading to OSC death. Several genes were differentially expressed in the ovarian tissue post-mortem, with the most enriched pathways being ferroptosis, fatty acid metabolism/biosynthesis, glutathione metabolism, citric acid cycle (TCA cycle), and arachidonic acid metabolism signaling pathways. Genes related to ferroptosis, such as vdac2, p53, and slc7a11, as well as metabolites such as adrenic acid and arachidic acid, can serve as reliable biomarkers for evaluating the viability of post-mortem OSCs. These findings provide valuable insights and theoretical support for the effective use of post-mortem GSCs and enhance strategies for germplasm resource conservation in fish.