Background Amphiprion ephippium is a significant ornamental marine species. Transportation stress, a common environmental stress during fish transport, may adversely affect their physiological immune systems.

Objective This study aims to investigate the biological characteristics of heat shock protein 8 (HSPA8) in A. ephippium and its expression patterns under transportation conditions.

Methods Using the liver tissue of A. ephippium, polymerase chain reaction (PCR) was employed to amplify and clone the coding sequences (CDS) of the HSPA8 gene. Bioinformatics analysis was conducted to evaluate its biological properties and structural features, along with detection of relative expression levels across different tissues. A transportation-induced stress model was constructed, and quantitative real-time PCR (qPCR) was used to monitor HSPA8 gene expression changes in various tissues at different transportation durations.

Results The CDS region of the HSPA8 gene in A. ephippium contained 1 953 bp, encoding 650 amino acid residues with a molecular weight of approximately 71.26 kDa and a theoretical isoelectric point of 5.22. The open reading frame (ORF) and protein sequence length of the HSPA8 gene in A. ephippium were identical to those of Amphiprion fish. The HSPA8 gene ORF of A. ephippium differed by 16 base pairs, with a sequence similarity of 99.18%, but the protein sequences were completely identical. Phylogenetic analysis showed that A. ephippium was closely related to the A. ocellaris. Subsequent analyses revealed the absence of signal peptide sequences and transmembrane domains, but it contained three typical structural domains of HSP70s proteins. qPCR analysis of HSPA8 gene tissue-specific expression demonstrated its presence in all 10 tested tissues (skin, muscle, spleen, gill, liver, heart, intestine, brain, kidney, and eye) . The gene showed highest relative expression in the eyes, followed by kidneys and brain tissues, with lower levels in heart and gill tissues, and the lowest level in skin. Under transport stress conditions, HSPA8 gene expression in the spleen reached highly significant upregulation at 6 and 12 hours, while intestinal upregulation was observed at 6 hours, with no other time points showing significance. Liver HSPA8 gene expression remained significantly lower than the control groups at 6, 12, 24, and 48 hours. Skin HSPA8 gene expression showed highly significant elevation at 24 hours compared to the control group, with no significant changes observed at other time points.

Conclusion The HSPA8 gene participates in immune tissue stress responses in A. ephippium, making it a reliable indicator of whether the species is experiencing a state of immunosuppression during stress. The above results provide a reference for further study of the immune regulatory function of HSPA8 under transport stress.