Analysis of the Expression Patterns of piRNAs in Response to Microsporidian Invasion in Midgut of Workers (Apis cerana cerana)

Piwi-interacting RNAs (piRNAs) play an essential part in transposon suppression, DNA methylation, and antiviral responses. The current understanding of the roles of piRNAs in honeybees is very limited. This study aims to analyze the expression pattern and regulatory role of piRNAs in the Asian honeybee (Apis cerana) responding to infection by Nosema ceranae, based on previously gained small RNA-seq data. Here, 450 and 422 piRNAs were respectively identified in the midgut tissues of Apis cerana cerana workers at 7 and 10 days post-inoculation (dpi) with N. ceranae, including 539 non-redundant ones. Additionally, one up-regulated (piR-ace-1216942) and one down-regulated (piR-ace-776728) piRNA were detected in the workers’ midgut at 7 dpi, targeting 381 mRNAs involved in 31 GO terms, such as metabolic processes, catalytic activity, and organelles, as well as 178 KEGG pathways, including lysosome, MAPK signaling pathway, and purine metabolism. A total of 35 up-regulated and 11 down-regulated piRNAs were screened from the workers’ midgut at 10 dpi, targeting 13,511 mRNAs engaged in 50 GO terms, such as biological regulation, transporter activity, and membrane, as well as 389 KEGG pathways, including the JAK-STAT signaling pathway, Hippo signaling pathway, and nitrogen metabolism. Further analysis indicated that 28 differentially expressed piRNAs (DEpiRNAs) in the midgut at 10 dpi could target 299 mRNAs annotated to three cellular immune pathways (lysosome, endocytosis, and phagosome), while 24 DEpiRNAs could target 205 mRNAs relevant to four humoral immune pathways (FoxO, JAK-STAT, NF-κB, and MAPK signaling pathway). Through Sanger sequencing and RT-qPCR, the expression of six randomly selected DEpiRNAs was verified. Moreover, the dual-luciferase reporter gene assay confirmed the binding relationships between piR-ace-446232 and CRT as well as between piR-ace-1008436 and EGFR. Our findings not only contribute to enrich our understanding of the role of piRNAs in honeybees but also provide a basis for exploring the host response to N. ceranae infection mediated by piRNAs.