Antibiotic Perturbation and Gut Symbiont Recolonization Affect the Cold Tolerance of Apis cerana

The gut microbiota contributes to host stress tolerance, yet its role in cold adaptation in the Eastern honey bee (Apis cerana) remains poorly understood. Here, A. cerana workers were treated with three antibiotics (tetracycline, streptomycin sulfate, and tylosin) at 50 μg/mL for 3 and 7 days, followed by cold survival assessment at 10 °C and 16S rRNA sequencing of the gut microbiota. Five cold tolerance-associated symbionts were subsequently evaluated in bacterial recolonization assays. Three days of antibiotic treatment significantly reduced bee survival under cold stress at 10 °C compared with the control, with the median survival time decreasing to 60 h across all groups. Seven days of streptomycin sulfate treatment further shortened the median survival time to 48 h. Antibiotics markedly depleted key symbionts, including Apibacter (from 23.89% to as low as 1.30%), Snodgrassella, and Lactobacillus. Recolonization with Apilactobacillus kunkeei and Gilliamella apicola significantly extended the cold median survival time to 92 h (p < 0.01 and p < 0.001, respectively), whereas Providencia rettgeri, Klebsiella oxytoca, and Bifidobacterium indicum extended it to 88 h. These findings indicate that gut symbionts modulate cold tolerance in A. cerana and that A. kunkeei and G. apicola can improve bee survival under low-temperature stress.