Comprehensive investigation and regulatory function of lncRNAs engaged in western honey bee larval immune response to Ascosphaera apis invasion

Ascosphaera apis is a fungal pathogen that exclusively infects bee larvae, causing chalkbrood disease, which results in severe damage for beekeeping industry. Long non-coding RNAs (lncRNAs) are versatile regulators in various biological processes such as immune defense and host-pathogen interaction. However, expression pattern and regulatory role of lncRNAs involved in immune response of bee host to A. apis invasion is still very limited. Here, Apis mellifera ligustica 4-, 5-, and 6-day-old larvae inoculated by A. apis spores (AmT1, AmT2, and AmT3 groups) and corresponding un-inoculated larval guts (AmCK1, AmCK2, and AmCK3 groups) were prepared and subjected to deep sequencing, followed by identification of lncRNAs, analysis of differentially expressed lncRNAs (DElncRNAs), and investigation of DElncRNA-regulated host immune responses. In total, 3 746 A. m. ligustica lncRNAs were identified, including 78 sense lncRNAs, 891 antisense lncRNAs, 1 893 intergenic lncRNAs, 346 bidirectional lncRNAs, and 210 intronic lncRNAs. Compared with corresponding un-inoculated larval guts, 357, 236, and 505 DElncRNAs were discovered in the A. apis-inoculated 4-, 5-, and 6-day-old larval guts. Additionally, there were 32 shared DElncRNAs. In AmCK1 vs AmT1, AmCK2 vs AmT2, and AmCK3 vs AmT3 comparison groups, 217, 129, and 272 DElncRNAs were respectively predicted to regulate neighboring genes via cis-acting manner, and these targets were associated with a series of GO terms and KEGG pathways of great importance, such as response to stimulus and Jak-STAT signal pathway. Moreover, competing endogenous RNA (ceRNA) network analysis indicated that 197, 95, and 356 DElncRNAs in the aforementioned three comparison groups could target 10, eight, and 21 DEmiRNAs and further target 147, 79, and 315 DEmRNAs, forming complex regulatory networks. Further investigation suggested that these targets were relevant to several key cellular and humoral immune pathways, such as phagosome and MAPK signaling pathway. Ultimately, the expression trends of nine randomly selected DElncRNAs were verified by RT-qPCR, indicative of the authenticity and reliability of our transcriptome data. Findings in this current work not only provide candidate DElncRNAs for functional study, but also lay a foundation for unclosing the mechanism underlying DElncRNA-regulated larval immune responses to A. apis infection.