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.
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