Bioscience Methods 2025, Vol.16, No.3, 162-172 http://bioscipublisher.com/index.php/bm 166 3.2 Core disease resistance-related genes discovered Cytokines play the role of signaling molecules in immune regulation. The IL-17 family members identified by Han et al. (2025a) are important proinflammatory cytokines, and their significant upregulation suggests that they play a key role in resisting bacterial infections. In particular, IL-17D and IL-17N subtypes increased dramatically in mucosal tissues when snakehead fish was infected with Nocardia, suggesting that they may recruit neutrophils and other antibacterial effects. In terms of signal transduction molecules, TNF receptor-associated factor (TRAF) is a connector molecule for many immune signaling pathways, such as TRAF6 involved in TLR and IL-1 receptor signals, and TRAF3 involved in antiviral RIG-I-like receptor signals (Chen et al., 2023). The eight snakehead fish TRAF genes cloned by Han et al. (2025b) provide a basis for studying the conservation and differences of these signaling pathways in fish immunity. Preliminary analysis showed that snakehead fish TRAF genes were widely expressed in different tissues and showed an upregulation trend after pathogen stimulation, suggesting that they were activated in the process of fish disease resistance. In terms of anti-infective effector molecules, some molecules that directly intervene in bactericidal and insecticidal activities have also been identified. For example, there are multiple antimicrobial peptide genes in the snakehead fish genome, such as β-defensin and hepcidin. Mucosal immunity-related genes are also worth mentioning, including the aforementioned polyimmunoglobulin receptor (pIgR) (Xu et al., 2025). snakehead fish pIgR is highly expressed in the intestinal mucosa and can bind to IgM/IgT and transport it to the surface. After infection, the expression of pIgR further increases, which helps to transport IgT antibodies generated in the mucosa to the infection site to neutralize pathogens. This shows that the immunity mediated by snakehead fish mucosal antibodies is similar to the IgA mechanism of mammals and is an important component of disease resistance. 3.3 Gene function verification strategy Traditional functional research methods include gene knockout, overexpression, gene mutation association analysis, etc. In model organisms such as zebrafish, gene knockout lines can be constructed to directly observe changes in mutant resistance to infection. However, for snakehead, there has been a lack of efficient gene manipulation methods, and functional verification mainly relies on in vitro cell experiments and heterologous models. One strategy is to overexpress or knock down candidate snakehead genes in bacteria or cell lines to evaluate their effects on pathogen growth. In recent years, CRISPR/Cas9 has been successfully used to functionally verify some important fish disease resistance genes. For example, the Nae1 gene of Atlantic salmon was knocked out in cell lines by CRISPR, and a significant decrease in IPNV replication was observed, thus confirming the role of the Nae1 gene (Pavelin et al., 2021). This method can also be used for snakehead research. With the development of snakehead primary cell and embryo manipulation technology, researchers can try to construct snakehead gene knockout lines or knockout individuals. Fortunately, Zhao et al. (2021) have reported the first successful case of snakehead gene editing: they used CRISPR/Cas9 to knock out the myostatin gene of spotted snakehead (Channa maculata) and cultivated genetic mutant fish with significantly accelerated growth. Gene overexpression and gene injection are also commonly used strategies. For example, the candidate gene mRNA is injected into the fertilized eggs of snakehead fish through microinjection, so that the gene is highly expressed in the fry stage, and then the effect is tested by pathogen challenge. 4 Application of Gene Editing Technology in Improving Disease Resistance of Snakehead Fish 4.1 Current status of CRISPR/Cas system and aquatic application CRISPR/Cas9 has simple design, low cost and high editing efficiency, and has become the preferred method for aquatic genome editing (Roy et al., 2022; Zhu et al., 2024). The application of gene editing in the aquatic field started with the model fish zebrafish and some economic fish. Around 2015, zebrafish successfully achieved CRISPR-mediated multi-site editing for the first time, laying the foundation for subsequent fish applications. Since then, gene editing attempts have been carried out in a number of farmed fish species, including Nile tilapia, carp, crucian carp, catfish, salmon, etc. (Zhu et al., 2024). So far, gene editing research has been reported in more
RkJQdWJsaXNoZXIy MjQ4ODYzNA==