Abstract
Ascosphaera apis is a fungal pathogen that specifically infects bee larvae, causing an outbreak of chalkbrood disease in the bee colony and a decline in the number of bee colonies. The role of miRNA regulation in honeybees in response to A. apis infection is unclear. In this study, based on small RNA-seq, we identified the differentially expressed miRNAs (DEmiRNAs) and their regulatory networks and functions in the gut of Apis cerana cerana on the first day (AcT1), the second day (AcT2) and the third day (AcT3) after A. apis infection, and analyzed the immune response mechanism of A. apis through the miRNAs-mRNA regulation network of A. apis infection. A total of 537 miRNAs were obtained, and 10, 27, and 54 DEmiRNAs were screened in the AcT1, AcT2, and AcT3 groups, respectively. The number of DEmiRNAs gradually increased with the infection time. Stem-loop RT-PCR results showed that most of the DEmiRNAs were truly expressed, and the expression trend of DEmiRNAs was consistent with the results of sRNA-seq. The top five GO terms of DEmiRNA-targeted mRNA were binding, cellular process, catalytic activity, metabolic process, and single-organism process. The main pathways enriched by KEGG were endocytosis, ubiquitin-mediated proteolysis, phagosome, and the JAK-STAT immune-related signaling pathways. The number of DEmiRNAs and target mRNAs of these related pathway genes increased with infection time. The miRNA-mRNA regulatory network analysis showed that ace-miR-539-y was the core miRNA of the early immune response in the gut of larvae infected with A. apis in the JAK-STAT pathway and phagosome, and ace-miR-1277-x was the core miRNA of the late immune response in the gut of larvae infected with A. apis in the JAK-STAT signaling pathway and phagosome. The results showed that miRNA participated in the immune response of honeybees to A. apis infection by regulating the host's energy metabolism, cellular immunity, and humoral immunity. The results of this study provide a basis for the regulation of miRNAs in A. c. cerana larvae in response to A. apis infection and provide new insights into host-pathogen interactions.