Abstract
Apoptosis is a genetically controlled process vital for homeostasis. This study examined the apoptotic response in the gut of Apis mellifera (A. mellifera) larvae to infection by Ascosphaera apis (A. apis) and its impact on host resistance and pathogen virulence. Here, Worker larvae of A. mellifera were inoculated with purified A. apis spores. We then quantified the expression of key apoptosis-related genes (AmCaspase-3, AmBax, and AmBcl-2) in the host gut and detected apoptotic cells via TUNEL assay. To functionally assess the role of apoptosis, larvae were treated with either the apoptosis inhibitor Z-VAD-FMK or the activator PAC-1, after which host survival, expression of apoptosis-associated genes, and the fungal virulence factor gene Ste11-like were analyzed. Our results showed that infection with A. apis significantly upregulated the expression of AmCaspase-3 and AmBax (p < 0.05) at 1-3 days post-inoculation (dpi), while the expression of AmBcl-2 was significantly reduced at 1 and 3 dpi (p < 0.05). Consistent with this, TUNEL assays revealed a markedly stronger green fluorescence signal in the guts of inoculated larvae at 3 dpi compared to uninfected controls, with clear co-localization of TUNEL and nuclear signals, confirming increased apoptosis. Pharmacological inhibition of apoptosis significantly enhanced the survival rate of A. apis-infected larvae, whereas apoptosis activation decreased larval survival. Accordingly, inhibiting apoptosis significantly suppressed the expression of AmCaspase-3 and AmBax (p < 0.001) and upregulated AmBcl-2 (p < 0.001). Conversely, apoptosis activation upregulated AmCaspase-3 (p > 0.05) and AmBax (p < 0.001), while significantly down-regulating AmBcl-2. Furthermore, apoptosis inhibition significantly down-regulated the fungal virulence gene Ste11-like, while its activation had the opposite effect. In summary, A. apis infection induces apoptosis in the larval gut by activating AmCaspase-3 and AmBax and suppressing AmBcl-2. Inhibiting this apoptotic response enhanced host survival by modulating the expression of host apoptosis-related genes and the fungal Ste11-like virulence factor. These findings provide new insights into the host response to A. apis and suggest a potential strategy for controlling chalkbrood disease.