Circular RNA response of Apis cerana cerana 6-day-old larvae to Ascosphaera apis stress

Abstract:

[Objective] Ascosphaera apis exclusively infects honeybee larvae, leading to chalkbrood, a fungal disease damaging honeybee health and beekeeping industry. The objective of this study was to investigate the differential expression pattern of circular RNAs (circRNAs) and differentially expressed circRNAs (DEcircRNAs) involved in Apis cerana cerana 6-day-old larval response to Ascosphaera apis stress, and the putative role of DEcircRNAs in host A. apis-response. [Methods] Normal and A. apis-infected 6-day-old larval guts of A. c. cerana (AcCK and AcT) were sequenced using linear RNA-removed circRNA-seq technology. CircRNAs were identified using find_circ software, followed by summary of length and circulization type of circRNAs. DEcircRNAs were screened out following the standard of |log₂(Fold change)|≥1 and P≤0.05. Source genes of DEcircRNAs were annotated to Gene Ontology (Go) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to gain function and pathway annotations. RT-qPCR was conducted to validate three randomly selected DEcircRNAs. [Results] Based on circRNA-seq, 76342570 and 68269362 raw reads were produced from AcCK and AcT, and 74524108 and 66974392 clean reads were obtained after strict quality control, with Q30 of 92.75% and 94% and GC content of 54.31% and 54.90%. In total, 23648400 anchor reads were mapped to the reference genome of Apis cerana. In AcCK and AcT, 805 and 702 circRNAs were identified; the lengths of these circRNAs were distributed among 201-1000 nt, and the most abundant circulization type was annotated exonic circRNA; however, the numbers of circRNAs distributed in various lengths and circulization types were different. There were 494 DEcircRNAs in AcCK vs AcT comparison group, including 257 up-regulated circRNAs and 237 down-regulated circRNAs, with novel_circ_000123 and novel_circ_000726 having the highest up-regulation and down-regulation levels. Source genes of the aforementioned DEcircRNAs were annotated to 11 biological process-associated terms, nine molecular function-associated terms, nine cellular component-associated terms, and 73 pathways. Further analysis demonstrated partial source genes were engaged in seven cellular immune pathways and three humoral immune pathways. [Conclusion] A. c. cerana 6-day-old larvae may response to A. apis stress by altering the numbers of circRNAs distributed in various lengths and circulization types, specifically expressing some circRNAs, and regulating the expression of partial circRNAs; several DEcircRNAs such as novel_circ_000027, novel_circ_000127 and novel_circ_000312 may play special roles in host A. apis-response via regulation of oxidative phosphorylation, cellular and humoral immune pathways. Our findings offered novel insights into further understanding molecular mechanisms underlying A. c. cerana larval response to A. apis and host-pathogen interaction.