Goldfish is a widely used model organism for studying various fields such as endocrinology, toxicology, molecular biology, and immunology. It serves as an excellent model for comparative immunology, allowing researchers to study the effects of immune stimuli on the fish immune system (Omori and Kon, 2019). This is crucial for understanding how different factors affect the immune system and its responses to various stimuli. To gain a deeper understanding of the goldfish immune system, investigating its innate immune system, antibacterial activity, and the molecular mechanisms involved is necessary. Aeromonas hydrophila, a Gram-negative bacterial species, can cause severe infectious diseases in aquaculture (Majumdar et al., 2006, Sreedharan et al., 2012). Previous studies have shown that A. hydrophila infection can lead to septicemia, ulcerative infections, tail rot, fin rot and death in goldfish (Rahman et al., 2001, Ren et al., 2017), as well as soft tissue infections, gastroenteritis and septicemia in humans (Banerjee et al., 2012). Thus, it is essential to understand the molecular defense mechanisms goldfish employ to protect themselves against A. hydrophila infection. By understanding these mechanisms, researchers can work towards developing new strategies to combat bacterial infections in both fish and humans.

Apoptosis, a type of programmed cell death, is an important component of the immune system and plays a pivotal role in responding to pathogenic invasions (Nagata, 2018). Morphologically, apoptosis is characterized by cell shrinkage, nuclear pyknosis and fragmentation (Huang et al., 2023). The process of apoptosis can be divided into two primary signaling pathways: the intrinsic pathway (mitochondrial pathway) and the extrinsic pathway (death receptor pathway), contingent upon the initiating stimuli. Caspases, a group of intracellular proteases, hold significant importance in both apoptosis and inflammation due to their highly conserved nature. These caspases can be further categorized into three subtypes based on their structure and role: initiator caspases (Casp2, 8, 9 and 10), executioner caspases (Casp3, 6 and 7), and inflammatory caspases (Casp1, 4, 5, 11, 12, 13 and 14) (Fu et al., 2019, Okun et al., 2006). Among these, Casp9 is a well-recognized activator caspase implicated in the promotion of the intrinsic apoptosis pathway. Structurally, Casp9 contains an N-terminal caspase recruitment domain (CARD domain) that interacts homotopically with the CARD domain of apoptotic protease activating factor 1 (Apaf-1) and the cytochrome-c released from the mitochondria, resulting in the formation of the apoptosome (Noori et al., 2021, Sahebazzamani et al., 2021). Additionally, the catalytic domain of Casp9 contains an auto-cleavage site located in the intersubunit-linker domain. This domain links the large subunit catalytic domain (p20) with the small subunit catalytic domain (p10) (Li et al., 2017). The apoptosome facilitates apoptosis by cleaving the catalytic domain of procasp9 into p35 and p12, subsequently activating executioner caspases (Casp3, 6 and 7) (Wu and Bratton, 2017, Wurstle et al., 2012).

Although apoptosis is a well-studied process in mammals, only a limited number of caspases have been identified and characterized in fish. Among these caspases, Casp9 has been implicated in intrinsic apoptotic pathways in higher vertebrates (Krumschnabel and Podrabsky, 2009), but its structure, function and role in lower vertebrates, including fish, remain poorly understood. However, studies have shown that Casp9 participates in the apoptosis process in several teleost species, such as Amphiprion clarkia (Udayantha et al., 2022), Danio rerio (Inohara and Nunez, 2000), Lateolabrax maculatus (Reis et al., 2007), Pseudosciaena crocea (Mu et al., 2010), Cyprinus carpio (Gao et al., 2013), Labeo rohita (Giri et al., 2018) and Bostrychus sinensis (Ding et al., 2020), in response to various pathogen-associated molecular patterns, to combat invading pathogens.

In the current study, we identified and cloned a Casp9 gene from goldfish. Subsequently, we investigated the subcellular localization of GfCasp9 in HEK293T cells and examined its over-expression to gain insights into its role in apoptosis. Furthermore, the study explored the immune function of GfCasp9 by analyzing its gene and protein expression patterns following A. hydrophila infection. The results of this study will enhance our understanding of the role of GfCasp9 in combating bacterial infections in teleosts.