Bombyx mori cypovirus (BmCPV) is one of the major viral pathogen for silkworm, and the genome of BmCPV is composed of 10 dsRNA segments. As construction system of recombinant BmCPV (rBmCPV) is scanty, researchers achieved little progress in studying gene function of BmCPV in recent decades. Here, 10 recombinant plasmids with a full-length cDNA of viral genome segments S1-S10 containing T7 promoter were constructed. After cotransfecting the BmN cells with the mixture of 10 in vitrotranscribed RNAs, pathological changes were observed. Real-time PCR and Western blot showed viral gene vp1 and structural proteins were expressed. It is found the genome of the rBmCPV is composed of 10 dsRNA segments similar to those of wild-type BmCPV. Moreover, viral particles and polyhedron with virions can be generated in the cotransfected cells and the injected silkworm midguts. These findings confirmed the formation of infective rBmCPV. Additionally, we found viable rBmCPV was generated by cotransfecting the mixture of in vitro-transcribed S1-S9 RNAs into the cultured cells, confirming polh was not essential for BmCPV replication. Moreover, an infectious rBmCPV expressing the DsRed protein was constructed based on this system. Further investigation showed S2 and S7 segments are indispensible for viral proliferation. Our findings demonstrated the construction system of rBmCPV can be utilized for exploring viral replication and pathogenesis, and investigated method for constructing rBmCPV will certainly facilitate developing novel biopesticides and expressing exogenous gene in the midgut of silkworm.

Honeybees are pivotal pollinators of crops and wild flora, and of great importance in supporting critical ecosystem balance. Nosema ceranae, a unicellular fungal parasite that infects midgut epithelial cells of honeybees, can dramatically reduce honeybee population and productivity. Here, midguts of Apis mellifera ligustica workers at 7 d and 10 d post inoculation (dpi) with sucrose solution (Ac7CK and Ac10CK) and midguts at 7 dpi and 10 dpi with sucrose solution containing N. ceranae spores (Ac7T and Ac10T) were sequenced using strand-specific cDNA library construction and next-generation sequencing. A total of 1956129858 raw reads were gained in this article, and after quality control, 1946489304 high-quality clean reads with a mean Q30 of 93.82% were obtained. The rRNA-removed clean reads were then aligned to the reference genome of Apis mellifera with TopHat2. For more insight please see “Genome-wide identification of long non-coding RNAs and their regulatory networks involved in Apis mellifera ligustica response to Nosema ceranae infection” [1]. Raw data were deposited in NCBI Sequence Read Archive (SRA) database under the BioProject number PRJNA406998. These data can be used for comparative analysis to identify differentially expressed coding RNAs and non-coding RNAs involved in host responses to N. ceranae stress, and for investigation of molecular mechanisms regulating N. ceranae-response.

Honeybees are susceptible to a variety of diseases, including chalkbrood, which is capable of causing huge losses of both the number of bees and colony productivity. This research is designed to characterize the transcriptome profiles of Ascosphaera apis-treated and un-treated larval guts of Apis mellifera ligustica in an attempt to unravel the molecular mechanism underlying the immune responses of western honeybee larval guts to mycosis. In this study, 24, 296 and 2157 genes were observed to be differentially expressed in A. apis-treated Apis mellifera (4-, 5- and 6-day-old) compared with un-treated larval guts. Moreover, the expression patterns of differentially expressed genes (DEGs) were examined via trend analysis, and subsequently, gene ontology analysis and KEGG pathway enrichment analysis were conducted for DEGs involved in up- and down-regulated profiles. Immunityrelated pathways were selected for further analysis, and our results demonstrated that a total of 13 and 50 DEGs were annotated in the humoral immune-related and cellular immune-related pathways, respectively. Additionally, we observed that many DEGs up-regulated in treated guts were part of cellular immune pathways, such as the lysosome, ubiquitin mediated proteolysis, and insect hormone biosynthesis pathways and were induced by A. apis invasion. However, more down-regulated DEGs were restrained. Surprisingly, a majority of DEGs within the Toll-like receptor signaling pathway, and the MAPK signaling pathway were up-regulated in treated guts, while all but two genes involved in the NF-κB signaling pathway were down-regulated, which suggested that most genes involved in humoral immune-related pathways were activated in response to the invasive fungal pathogen. This study's findings provide valuable information regarding the investigation of the molecular mechanism of immunity defenses of A. m. ligustica larval guts to infection with A. apis.

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】环状RNA（circular RNA，circRNA）在可变剪接、转录调控和来源基因的表达调控等方面具有重要功能。本研究旨在探究意大利蜜蜂（Apis mellifera ligustica，简称意蜂）工蜂中肠发育过程中circRNA的表达谱及其发育过程中的差异表达circRNA（differentially expressed circRNA,DEcircRNA），进而在转录组水平探究DEcircRNA在中肠发育中的作用。【方法】基于前期获得的意蜂7和10日龄工蜂中肠样品（Am7和Am10）的全转录组数据，利用find_circ软件从质控后的数据中预测circRNA。采用RPM算法归一化处理得到circRNA的表达量。利用DESeq软件对circRNA进行差异分析，按照差异倍数（fold change）≥2、P＜0.05及错误发现率（false discovery rate，FDR）＜0.05条件筛选DEcircRNA。通过BLAST比对GO和KEGG数据库，对DEcircRNA的来源基因进行功能和代谢通路注释。利用TargetFinder软件预测DEcircRNA-miRNA及DEcicRNA-miRNA-mRNA调控网络，通过Cytoscape v.3.2.1软件对调控网络进行可视化。通过实时荧光定量PCR（Real-time quantitative PCR,RT-qPCR）验证测序数据的可靠性。【结果】意蜂中肠各样品比对上参考基因组的短序列读段数平均为19 616 356条。Am7与Am10的组内Pearson相关系数均在0.950及以上。共预测出256个DEcicRNA，包括105个上调circRNA和151个下调circRNA。Novel_circ_009675和novel_circ_013879分别在Am7和Am10中高量表达。DEcircRNA的来源基因可注释到包括结合、单组织进程及细胞进程在内的32个GO条目，其中分别有35、35和7个来源基因注释到催化活性、代谢进程和应激反应。上述来源基因还可注释到35条KEGG代谢通路，其中分别有5、5和4个来源基因注释到Hippo信号通路、内吞作用和吞噬体；进一步分析发现分别有1、2和2个来源基因注释到磷酸肌醇代谢、淀粉和蔗糖代谢和半乳糖代谢等物质代谢通路，5、4、3、1和1个注释到内吞作用、吞噬体、溶酶体、泛素介导的蛋白水解和MAPK信号通路等免疫通路。上述结果表明相应的DEcircRNA广泛参与意蜂工蜂中肠的生长发育、新陈代谢和免疫防御。DEcicRNA-miRNA调控网络分析结果显示，141个DEcircRNA可靶向结合107个miRNA，其中多数仅能结合1-2个miRNA，但novel_circ_011577和novel_circ_010719结合的靶miRNA数可达32和28个；此外，mir-136-y、ame-miR-6001-3p及mir-136-y结合的circRNA数最多，分别为15、14和14个；表明相应的DEcircRNA可作为竞争性内源RNA在意蜂中肠发育过程发挥作用。进一步构建DEcicRNAs-ame-miR-6001-3p-mRNA调控网络，分析结果显示14个DEcicRNA可共同靶向结合ame-miR-6001-3p，表明它们可能通过调控ame-miR-6001-3p对意蜂中肠干细胞的分裂和分化进行间接调控。随机选取6个DEcricRNA进行RT-qPCR验证，结果显示5个的表达量变化趋势与测序结果一致，证实了本研究测序结果的可靠性。【结论】通过对意蜂工蜂中肠发育过程中的DEcircRNA深入分析，提供了circRNA在意蜂工蜂中肠发育过程中的表达谱和差异表达信息，揭示了DEcircRNA在中肠发育过程中的作用，为中肠发育相关的关键circRNA的筛选和功能研究打下了基础。