Data_Sheet_1_In-depth investigation of microRNA-mediated cross-kingdom regulation between Asian honey bee and microsporidian.xlsx

Asian honey bee Apis cerana is the original host for Nosema ceranae, a unicellular fungal parasite that causes bee nosemosis throughout the world. Currently, interaction between A. cerana and N. ceranae is largely unknown. Our group previously prepared A. c. cerana workers’ midguts at 7 days post inoculation (dpi) and 10 dpi with N. ceranae spores as well as corresponding un-inoculated workers’ midguts, followed by cDNA library construction and a combination of RNAs-seq and small RNA-seq. Meanwhile, we previously prepared clean spores of N. ceranae, which were then subjected to cDNA library construction and deep sequencing. Here, based on the gained high-quality transcriptome datasets, N. ceranae differentially expressed mRNAs (DEmiRNAs) targeted by host DEmiRNAs, and A. c. cerana DEmRNAs targeted by microsporidian DEmiRNAs were deeply investigated, with a focus on targets involved in N. ceranae glycolysis/glyconeogenesis as well as virulence factors, and A. c. cerana energy metabolism and immune response. In A. c. cerana worker’s midguts at 7 (10) dpi (days post inoculation), eight (seven) up-regulated and six (two) down-regulated miRNAs were observed to target 97 (44) down-regulated and 60 (15) up-regulated N. ceranae mRNAs, respectively. Additionally, two up-regulated miRNAs (miR-60-y and miR-676-y) in host midgut at 7 dpi could target genes engaged in N. ceranae spore wall protein and glycolysis/gluconeogenesis, indicating potential host miRNA-mediated regulation of microsporidian virulence factor and energy metabolism. Meanwhile, in N. ceranae at 7 (10) dpi, 121 (110) up-regulated and 112 (104) down-regulated miRNAs were found to, respectively, target 343 (247) down-regulated and 138 (110) down-regulated mRNAs in A. c. cerana workers’ midguts. These targets in host were relevant to several crucial cellular and humoral immune pathways, such as phagasome, endocytosis, lysosomes, regulation of autophagy, and Jak–STAT signaling pathway, indicative of the involvement of N. ceranae DEmiRNAs in regulating these cellular and humoral immune pathways. In addition, N. ceranae miR-21-x was up-regulated at 7 dpi and had a target relative to oxidative phosphorylation, suggesting that miR-21-x may be used as a weapon to modulate this pivotal energy metabolism pathway. Furthermore, potential targeting relationships between two pairs of host DEmiRNAs-microsporidian DEmRNAs and two pairs of microsporidian DEmiRNAs-host DEmRNAs were validated using RT-qPCR. Our findings not only lay a foundation for exploring the molecular mechanism underlying cross-kingdom regulation between A. c. cerana workers and N. ceranae, but also offer valuable insights into Asian honey bee-microsporidian interaction.

东方蜜蜂（Apis cerana）是东方蜜蜂微孢子虫（Nosema ceranae）的原始宿主，后者是一种单细胞真菌类寄生虫，在全球范围内引发蜜蜂微孢子虫病。目前，东方蜜蜂与东方蜜蜂微孢子虫之间的互作机制仍鲜为人知。本课题组前期分别制备了接种东方蜜蜂微孢子虫孢子后7天（days post inoculation，缩写dpi）、10天的中华蜜蜂（Apis cerana cerana, A. c. cerana）工蜂中肠样本，以及未接种的对应对照组中肠样本；随后构建了cDNA文库，并联合进行了RNA测序（RNA-seq）和小RNA测序（small RNA-seq）。同时，本课题组前期纯化获得了东方蜜蜂微孢子虫的纯净孢子，随后对其构建cDNA文库并进行深度测序。本研究基于所获得的高质量转录组数据集，深入分析了宿主差异表达miRNA（differentially expressed microRNAs, DEmiRNAs）靶向的东方蜜蜂微孢子虫差异表达mRNA，以及微孢子虫差异表达miRNA靶向的中华蜜蜂差异表达mRNA；重点关注了参与东方蜜蜂微孢子虫糖酵解/糖异生通路、毒力因子的靶标，以及中华蜜蜂能量代谢与免疫应答相关的靶标。在接种后7（10）天的中华蜜蜂工蜂中肠中，分别有8（7）个上调表达、6（2）个下调表达的miRNA，靶向97（44）个下调表达及60（15）个上调表达的东方蜜蜂微孢子虫mRNA。此外，接种后7天宿主中肠内的2个上调表达miRNA（miR-60-y与miR-676-y），可靶向东方蜜蜂微孢子虫的孢子壁蛋白及糖酵解/糖异生相关基因，提示宿主miRNA可能介导对微孢子虫毒力因子与能量代谢的调控。同时，在接种后7（10）天的东方蜜蜂微孢子虫中，分别有121（110）个上调表达、112（104）个下调表达的miRNA，靶向中华蜜蜂工蜂中肠内的343（247）个下调表达及138（110）个下调表达的mRNA。宿主中的这些靶标涉及多条关键的细胞免疫与体液免疫通路，例如吞噬体（phagasome）、胞吞作用（endocytosis）、溶酶体（lysosomes）、细胞自噬调控以及JAK-STAT信号通路（Jak–STAT signaling pathway），这表明东方蜜蜂微孢子虫的差异表达miRNA参与调控这些细胞与体液免疫通路。此外，东方蜜蜂微孢子虫的miR-21-x在接种后7天呈上调表达，其靶标与氧化磷酸化（oxidative phosphorylation）通路相关，提示miR-21-x可作为调控该关键能量代谢通路的效应分子。进一步地，本研究通过实时荧光定量PCR（reverse transcription quantitative PCR, RT-qPCR）验证了2对宿主DEmiRNA-微孢子虫DEmRNA以及2对微孢子虫DEmiRNA-宿主DEmRNA的潜在靶向关系。本研究结果不仅为解析中华蜜蜂工蜂与东方蜜蜂微孢子虫之间的跨王国调控分子机制奠定了基础，同时也为深入理解东方蜜蜂-微孢子虫的互作关系提供了重要参考。