Ascosphaera apis is an obligate fungal pathogen of honeybee larvae that leads to chalkbrood, which causes heavy losses for the apiculture in China and many other countries. In this article, guts of 4-, 5-, 6-day-old Apis mellifera ligustica larvae challenged by A. apis (AmT1, AmT2, AmT3) and normal 4-day-old larval guts (AmCK) were sequenced using next-generation sequencing technology. On average, 29196197, 28690943, 29779715 and 30496725 raw reads were yielded from these four groups; an average of 29540895 clean reads were obtained after quality control. In addition, the mapping ratio of clean reads in treatment and control groups to the Apis mellifera genome were over 97.16%. For more insight please see “Uncovering the immune responses of Apis mellifera ligustica larval gut to Ascosphaera apis infection utilizing transcriptome sequencing” [1]. The raw data were submitted to the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession numbers: SRR4084091, SRR4084092, SRR4084095, SRR4084096, SRR4084097, SRR4084098, SRR4084099, SRR4084100, SRR4084101, SRR4084102, SRR4084093, SRR4084094.

Currently, knowledge of circular RNAs (circRNAs) in insects including honeybee is extremely limited. Here, differential expression profiles and regulatory networks of circRNAs in the midguts of Apis cerana cerana workers were comprehensively investigated using transcriptome sequencing and bioinformatics. In total, 9589 circRNAs (201–800 nt in length) were identified from 8-day-old and 11-day-old workers’ midguts (Ac1 and Ac2); among them, 5916 (61.70%) A. cerana cerana circRNAs showed conservation with our previously indentified circRNAs in Apis mellifera ligucstica workers’ midguts (Xiong et al., Acta Entomologica Sinica 61:1363–1375, 2018). Five circRNAs were confirmed by RT-PCR and Sanger sequencing. Interestingly, novel_circ_003723, novel_circ_002714, novel_circ_002451, and novel_circ_001980 were highly expressed in both Ac1 and Ac2. In addition, the source genes of circRNAs were involved in 34 GO terms including organelle and cellular process and 141 pathways such as endocytosis and Wnt signaling pathway. Moreover, 55 DEcircRNAs including 34 upregulated and 21 downregulated circRNAs were identified in Ac2 compared with Ac1. circRNA-miRNA regulatory networks indicated that 1060 circRNAs can target 74 miRNAs; additionally, the DEcircRNA-miRNA-mRNA networks suggested that 13 downregulated circRNAs can bind to eight miRNAs and 29 miRNA-targeted mRNAs, while 16 upregulated circRNAs can link to 9 miRNAs and 29 miRNA-targeted mRNAs. These results indicated that DEcircRNAs as ceRNAs may play a comprehensive role in the growth, development, and metabolism of the worker’s midgut via regulating source genes and interacting with miRNAs. Notably, eight DEcircRNAs targeting miR-6001-y were likely to be key participants in the midgut development. Our findings not only offer a valuable resource for further studies on A. cerana cerana circRNA and novel insights into understanding the molecular mechanisms underlying the midgut development of eastern honeybee but also provide put

【目的】前期已对意大利蜜蜂（Apis mellifera ligustica，简称意蜂）7日龄工蜂中肠（Am7）、10日龄工蜂中肠（Am10）进行全转录组测序，基于高质量的组学数据探究中肠发育过程中的差异基因表达谱及其调控网络，以期解析意蜂工蜂中肠发育的分子机理。【方法】根据FPKM（fragments per kilobase of transcript per million mapped reads）算法计算基因表达量，并以|log2 fold change|≥1且P≤0.05作为标准筛选得到差异表达基因（differentially expressed gene，DEG）。利用TargetFinder软件预测ame-miR-6001-3p的靶mRNA。利用相关生物信息学软件，对全部DEG进行GO和KEGG数据库注释。筛选出与AMPK、P13K-Akt、Wnt、cAMP、FoxO、Hippo、mTOR、Jak-STAT、Toll-like受体、TGF-beta、Notch、MAPK和NF-κB等13条信号通路存在富集关系的DEG，以及与ame-miR-6001-3p存在靶向结合关系的DEG，通过Cytoscape软件构建调控网络将其富集关系与调控关系可视化。利用茎环反转录PCR（Stem loop RT-PCR）和实时荧光定量PCR（RT-qPCR）验证ame-miR-6001-3p以及DEG在Am7和Am10中的差异表达情况。【结果】Am7 vs Am10比较组中共有1 038个DEG，包括515个上调基因和523个下调基因。这些DEG涉及细胞进程、代谢进程和催化活性等功能条目，并显著富集在氧化磷酸化、氨基糖与核苷酸糖代谢、脂肪酸代谢和嘌呤代谢等能量和物质代谢通路，表明工蜂中肠内存在旺盛细胞生命与新陈代谢活动。表达量聚类分析发现分别有20、18、15和14个DEG富集在 AMPK信号通路、P13K-Akt信号通路、内吞作用和Hippo信号通路。57个DEG与P13K-Akt、Wnt、Jak-STAT等上述13条与生长发育和免疫防御相关的信号通路存在富集关系，且1个DEG可与多条信号通路存在富集关系。调控网络分析结果显示，分别有54个上调基因和44个下调基因可被ame-miR-6001-3p靶向结合；上调基因富集在磷酸肌醇代谢、胰岛素信号通路、Hippo信号通路和谷胱甘肽代谢等43条代谢通路，而下调基因富集在Hippo信号通路、新陈代谢途径、谷胱甘肽代谢和花生四烯酸代谢等20条代谢通路。RT-qPCR结果显示随机挑选的6个DEG的表达量变化趋势与测序数据一致，证实了本研究中基因差异表达真实可靠。此外ame-miR-6001-3p在Am7和Am10内均真实表达，并在Am10中的相对表达量显著较低。【结论】对意蜂工蜂中肠发育过程的DEG表达谱和DEG与ame-miR-6001-3p之间的调控关系，以及DEG的潜在作用进行深入分析和探讨，发现DEG可参与TGF-beta、Wnt、Hippo、Notch、PI3K-Akt、mTOR、AMPK和NF-κB等各类信号通路进而影响中肠的生长发育和免疫防御，DEG可通过与显著下调表达的ame-miR-6001-3p形成复杂的调控网络参与意蜂工蜂中肠发育过程中胰岛素信号通路等多条代谢途径。

N osema ceranae is a widespread fungal pathogen of honeybees, which is infective to all castes in the colony, including queens, drones and workers. Nosemosis caused by N. ceranae poses a big challenge for apiculture all over the world. In this articleHere, midguts of normal and N. ceranae-infected Apis cerana cerana workers at 7 (10) days post infection (dpi) were sequenced utilizing small RNA sequencing (sRNA-seq). Totally, 150.54 Mb raw reads were produced in this article, and 144.26 Mb high-quality clean reads with a mean ratio of 95.83% were obtained after strict filtering and quality control. For more insight please see “Comparative identification of microRNAs in Apis cerana cerana workers' midguts responding to Nosema ceranae invasion” [1]. Raw data are available in NCBI Sequence Read Archive (SRA) database under the BioProject number PRJNA487111. Our data can be used for investigating differentially expressed microRNAs (miRNAs) and piRNAs and their regulatory roles engaged in A. c. cerana response to N. ceranae infection, and for offering potential candidates for uncovering the molecular mechanisms regulating eastern honeybee-microsporidian interactions.

Chalkbrood is the most common fungal disease in honeybees. The objective of this study was to reveal immune responses in the Apis cerana cerana larval gut following Ascosphaera apis invasion. Combining a previously assembled transcriptome of A. c. cerana larval gut and the high-throughput sequencing data obtained in this study, 6152 differentially expressed genes (DEGs) were clustered into eight profiles. Trend analysis showed three significant up-regulated profiles (p ≤ 0.05) and three down-regulated profiles. Gene Ontology (GO) term analysis suggested that DEGs within significant up-regulated and down-regulated clusters were enriched in 46 and 38 functional groups, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated a majority of DEGs were involved in ribosome structure or function, carbon metabolism, biosynthesis of amino acids, and oxidative phosphorylation. In addition, 142 and 14 DEGs were annotated in the cellular immune- and humoral immune-related pathways, respectively. Further investigation indicated that DEGs up-regulated in cellular immune and humoral immune pathways outnumbered those that were downregulated. Moreover, immune responses of A. c. cerana and Apis mellifera ligustica larvae were compared and studied to decipher resistance of eastern honeybee larvae to A. apis. These results demonstrated that a large number of genes involved in immunity-related pathways were activated by A. apis. Our findings provided valuable information for elucidating the molecular mechanisms underlying immune responses of A. c. cerana larvae to A. apis infection and pathogen-host interactions during chalkbrood infection.