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

Abstract

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, the gut tissues of 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 competing endogenous RNA (ceRNA) network. 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. In the 4-, 5-, and 6- comparison groups, 357, 236, and 505 DElncRNAs were discovered. Additionally, 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 signaling pathway. Moreover, 197, 95, and 356 DElncRNAs were observed to 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 engaged in 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, confirming 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 invasion.

Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae

“…In another previous works ( Guo et al, 2019a , b ), the A. m. ligustica worker 3-day-old larvae ( n = 9) in treatment group were fed 50 μL of artificial diet containing A. apis spores, with a final concentration of 1 × 10 7 spores/mL; while the 3-day-old larvae ( n = 9) in control group were fed 50 μL of artificial diet without fungal spores; the larvae in treatment and control groups were reared in two chambers under the conditions of 35.0 ± 0.5°C and 90% RH (relative humidity); the diets were replaced with new diets without spores every 24 h; the 4-, 5-, and 6-day-old larval guts were, respectively, dissected according to our established protocol ( Guo et al, 2019c ), nine gut tissue for each day-old; the prepared guts were quickly frozen in liquid nitrogen and then conserved at −80°C; the aforementioned experiments were repeated in triplicate; these gut samples were subjected to RNA isolation, cDNA library construction, and next-generation sequencing on the Illumina MiSeq™ system by Guangzhou Genedenovo Biotechnology Co., Ltd. ( Ye et al, 2022 ). The raw data are available in the NCBI SRA database under the BioProject number PRJNA565629.…”

Systematic Characterization and Regulatory Role of lncRNAs in Asian Honey Bees Responding to Microsporidian Infestation

“…According to the method described by Ye et al [ 64 ], neighboring genes within 10 kb upstream and downstream of DElncRNAs were surveyed and considered as potential targets. Subsequently, using the Blast tool in the NCBI database ( , accessed on 5 September 2022), the neighboring genes were, respectively, aligned to the GO ( , accessed on 5 September 2022) and KEGG ( , accessed on 5 September 2022) databases to the gain corresponding functional and pathway annotations.…”

Novel Insights into the circRNA-Modulated Developmental Mechanism of Western Honey Bee Larval Guts

“…Previously, the worker larval gut samples of A. m. ligustica, a subspecies of A. mellifera widely used in the beekeeping industry, were prepared followed by the deep sequencing of cDNA libraries [32]. Our data could provide not only a new perspective into the honey bee gut development but also a foundation for elucidating the developmental mechanism of the larval guts.…”