International Journal of Molecular Zoology, 2025, Vol.15, No.2, 48-57 http://animalscipublisher.com/index.php/ijmz 53 hypoxia. Combining transcriptome and metabolome analysis can correspond changes in gene expression to changes in metabolic flux, revealing the regulatory mechanism in more depth. 5.2 CRISPR/Cas9-mediated functional gene knockout experiments to verify key sites Functional genomics, especially CRISPR/Cas9 gene editing technology, provides a powerful tool for verifying the key genes and sites of catfish hypoxia tolerance. Although there are relatively few gene knockout experiments in aquatic fish such as catfish, studies on other model fish have shown that targeted knockout of hypoxia-responsive genes can significantly affect the hypoxia-tolerant phenotype. For example, HIF-1α and HIF-3α in zebrafish are core transcription factors that regulate hypoxia response. By knocking out the zebrafish hif-3α gene through CRISPR/Cas9, researchers observed that the mutant fish had impaired erythropoiesis and reduced hemoglobin, resulting in a significantly shortened survival time in hypoxic water. This result directly proves the functional importance of HIF-3α to fish hypoxia tolerance (Cai et al., 2020). Similarly, site-directed mutagenesis of the zebrafish pVHL gene can simulate the natural variation of plateau fish pVHL. The results showed that zebrafish with plateau mutations showed stronger HIF signaling activity and hypoxia tolerance. This shows that mutations at certain key sites on the pVHL protein can improve HIF stability, thereby enhancing hypoxia adaptability (Chen et al., 2020). 5.3 Phenotype-gene association analysis under simulated hypoxia experimental system Phenotype-gene association analysis under simulated hypoxic environment provides an effective path to reveal the complex traits of catfish hypoxia tolerance. It is not limited to single gene effects, but captures the combined effects of multiple genes and pathways, which is closer to the real biological situation. With the application of more high-resolution molecular markers and the testing of larger population samples, it is expected to depict the genetic architecture map of catfish hypoxia tolerance traits and identify markers and sites that can be used for molecular breeding (Yu et al., 2021). It is reported that when channel catfish are exposed to hypoxia at night, the expression of genes related to feeding and stress in their hypothalamus changes in circadian rhythm, affecting feeding behavior and physiological state. This transcriptional response to environmental changes is also related to the individual's genotype: some individuals may have "pre-adaptive" gene expression regulation, which prepares them before the onset of hypoxia, thereby showing stronger tolerance. This hypothesis can be tested by comparing the expression profiles of individuals with different tolerance. 6 Case Analysis of the Evolution of Catfish's Tolerance to Hypoxia 6.1 Study on the evolution of hypoxia tolerance in representative catfish species Catfish are a group with extremely high species diversity, and several representative species are known for their excellent tolerance to hypoxia, making them ideal models for studying hypoxia adaptation. For example, the American channel catfish (Ictalurus punctatus, commonly known as "channel catfish" in Chinese) is an important species for freshwater aquaculture in North America. It can survive in high-density aquaculture ponds with significantly reduced dissolved oxygen at night. This ecological success is attributed to the channel catfish's strong hypoxia-tolerant genotype: studies have pointed out that the reason why channel catfish is widely cultured is partly because it has a "relatively high tolerance to hypoxia" compared to other catfish (Yang et al., 2018). The walking catfish in Asia (such as Clarias batrachus, commonly known as "crawling catfish") is also known for its ability to "walk" on land. They often live in low-oxygen rice fields and shallow ponds. When the original water body dries up, they can rely on their pectoral fins to support their bodies and crawl forward to find new water sources. Walking catfish has the ability to breathe through gills and air through suprapharyngeal organs, and is a "double champion" in tolerance to hypoxia and drought. Its whole genome sequencing study revealed that this fish has particularly amplified genes related to oxygen storage and utilization (such as myoglobin), and has adapted to terrestrial life by enhancing hemoglobin expression and angiogenesis (Li et al., 2018). In addition, other catfish species, such as the armored catfish in South America, also show tolerance to hypoxia, and they can use the intestine to breathe air in hypoxic rivers. Physiological studies on these species have shown that even if the strategies in heart rate, ventilation and other responses are different, their adaptation principles of increasing oxygen uptake and reducing oxygen consumption are consistent (Scott et al., 2017).
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