Global discovery, expression pattern, and regulatory role of miRNAlike RNAs in Ascosphaera apis infecting the Asian honeybee larvae

Xiaoyu Liu ¹ Sihai Geng ^1,2,3 Daoyou Ye ¹ Wenhua Xu ¹ Yidi Zheng ¹ Fangji Wang ¹ Jianpeng Lei ¹ Ying Wu ⁴ Haibin Jiang ⁴ Ying Hu ¹ Chen Dafu ^1,2,3 Tizhen Yan ⁵ Rui Guo ^1,2,3* Jian Feng Qiu ^1,2,3*

• ¹ Fujian Agriculture and Forestry University, Fuzhou, China
• ² National & Local United Engineering Laboratory of Natural Biotoxin, Fuzhou 350002, China, Fuzhou, China
• ³ Apitherapy Research Institute of Fujian Province, Fuzhou, China
• ⁴ Apiculture Science Institute of Jinlin province, Jilin City, Jilin Province, China
• ⁵ Dongguan Maternal and Child Health Hospital, Guangdong, China

Ascosphaera apis, a specialized fungal pathogen, causes lethal infection in honeybee larvae. miRNAlike small RNAs (milRNAs) are fungal small non-coding RNAs similar to miRNAs, which have been shown to regulate fungal hyphal growth, spore formation, and pathogenesis. Based on the transcriptome data, differentially expressed miRNA-like RNAs (DEmilRNAs) in A. apis infecting the Apis cerana cerana worker 4-, 5-, and 6-day-old larvae (Aa-4, Aa-5, and Aa-6) were screened and subjected to trend analysis, followed by target prediction and annotation as well as investigation of regulatory networks, with a focus on sub-networks relative to MAPK signaling pathway, glycerolipid metabolism, superoxide dismutase, and enzymes related to chitin synthesis and degradation. A total of 606 milRNAs, with a length distribution ranging from 18 nt to 25 nt, were identified. The first nucleotide of these milRNAs presented a bias towards U, and the bias patterns across bases of milRNAs were similar in the aforementioned three groups. There were 253 milRNAs, of which 68 up-and 54 down-regulated milRNAs shared by these groups. Additionally, the expression and sequences of three milRNAs were validated by stem-loop RT-PCR and Sanger sequencing. Trend analysis indicated that 79 DEmilRNAs were classified into three significant profiles (Profile4, Profile6, and Profile7). Target mRNAs of DEmilRNAs in these three significant profiles were engaged in 42 GO terms such as localization, antioxidant activity, and nucleoid. These targets were also involved in 120 KEGG pathways including lysine biosynthesis, pyruvate metabolism, and biosynthesis of antibiotics. Further investigation suggested that DEmilRNA-targeted mRNAs were associated with the MAPK signaling pathway, glycerolipid metabolism, superoxide dismutase, and enzymes related to chitin synthesis and degradation.Moreover, the binding relationships between aap-milR10516-x and ChsD as well as between aap-milR-2478-y and mkh1 were confirmed utilizing a combination of dual-luciferase reporter gene assay and RT-qPCR. Our data not only provide new insights into the A. apis proliferation and invasion, but also lay a basis for illustrating the DEmilRNA-modulated mechanisms underlying the A. apis infection.