Apoptosis and Relevant Genes Are Engaged in the Response of Apis mellifera Larvae to Ascosphaera apis Invasion

Apoptosis is a genetically controlled process vital for homeostasis. This study examined the apoptotic response in the gut of <i>Apis mellifera</i> (<i>A. mellifera</i>) larvae to infection by <i>Ascosphaera apis</i> (<i>A. apis</i>) and its impact on host resistance and pathogen virulence. Here, Worker larvae of <i>A. mellifera</i> were inoculated with purified <i>A. apis</i> spores. We then quantified the expression of key apoptosis-related genes (<i>AmCaspase</i>-3, <i>AmBax</i>, and <i>AmBcl</i>-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 <i>Ste</i>11-<i>like</i> were analyzed. Our results showed that infection with <i>A. apis</i> significantly upregulated the expression of <i>AmCaspase</i>-3 and <i>AmBax</i> (<i>p</i> < 0.05) at 1-3 days post-inoculation (dpi), while the expression of <i>AmBcl</i>-2 was significantly reduced at 1 and 3 dpi (<i>p</i> < 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 <i>A. apis</i>-infected larvae, whereas apoptosis activation decreased larval survival. Accordingly, inhibiting apoptosis significantly suppressed the expression of <i>AmCaspase</i>-3 and <i>AmBax</i> (<i>p</i> < 0.001) and upregulated <i>AmBcl</i>-2 (<i>p</i> < 0.001). Conversely, apoptosis activation upregulated <i>AmCaspase</i>-3 (<i>p</i> > 0.05) and <i>AmBax</i> (<i>p</i> < 0.001), while significantly down-regulating <i>AmBcl</i>-2. Furthermore, apoptosis inhibition significantly down-regulated the fungal virulence gene <i>Ste</i>11-<i>like</i>, while its activation had the opposite effect. In summary, <i>A. apis</i> infection induces apoptosis in the larval gut by activating <i>AmCaspas</i>e-3 and <i>AmBax</i> and suppressing <i>AmBcl</i>-2. Inhibiting this apoptotic response enhanced host survival by modulating the expression of host apoptosis-related genes and the fungal <i>Ste</i>11-<i>like</i> virulence factor. These findings provide new insights into the host response to <i>A. apis</i> and suggest a potential strategy for controlling chalkbrood disease.