Regulatory Manner and Role of Circular RNAs in the Microsporidian Invasion of Asian Honeybee

Vairimorpha ceranae, an obligate intracellular parasitic fungus, infects the midgut of worker Apis cerana cerana, disrupts nutritional metabolism and immune homeostasis of hosts, and poses severe threats to honeybee health and the sustainable development of apiculture. To date, the expression dynamics of circular RNAs (circRNAs) during pathogen infection and their regulatory functions as competing endogenous RNAs (ceRNAs) remain poorly characterized. In this study, circRNA sequencing was performed on purified V. ceranae spores (VcCK), as well as the midguts of A. cerana cerana worker bees at 7 days post-infection (VcT1) and 10 days post-infection (VcT2). A total of 8,986,470 and 315 circRNAs were identified from the three groups, with their lengths predominantly ranging from 200 to 600 nt. Differential expression analysis screened 575 and 594 differentially expressed circRNAs (DEcircRNAs) from the VcCK vs. VcT1 and VcCK vs. VcT2 comparison groups, respectively. The host genes generating these DEcircRNAs were annotated to 316 and 310 GO terms, alongside 167 KEGG pathways. CeRNA network analysis revealed that DEcircRNAs including novel_circ_006653 target multiple miRNAs, and their downstream target mRNAs are significantly enriched in energy metabolism pathways such as carbon metabolism and glycolysis/gluconeogenesis. RT-qPCR validation exhibited high consistency with transcriptome sequencing data. Further analyses demonstrated that V. ceranae infection drastically remodels the circRNA expression landscape: the total number of circRNAs was sharply reduced, and the dominant circRNA subtype shifted from antisense circRNAs to single-exon circRNAs. Several DEcircRNAs (novel_circ_006653, novel_circ_004839, novel_circ_005596, etc.) may participate in pathogen infection progression via the ceRNA regulatory axis by modulating host energy metabolism, immune responses and cellular biological processes. This study provides novel insights into the molecular interaction mechanisms between microsporidian parasites and their honeybee hosts, and identifies potential molecular targets for the prevention and control of microsporidiosis.