Ascospheara apis is a widespread fungal pathogen that exclusively invades honeybee larvae. Thus far, non-coding RNA in A. apis has not yet been documented. In this study, we sequenced A. apis using strand specific cDNA library construction and Illumina RNA sequencing methods, and identified 379 lncRNAs, including antisense lncRNAs, lincRNAs, intronic lncRNAs and sense lncRNAs. Additionally, these lncRNAs were found to be shorter in length and have fewer exons and transcript isoforms than protein-coding genes, similar to those identified in mammals and plants. Furthermore, the existence of 15 predicted lncRNAs of A. apis was confirmed using RT-PCR and expression levels of 11 were lower than those of adjacent protein-coding genes. Our findings not only enlarge the lncRNA database for fungi, but also lay a foundation for further investigation of potential lncRNAmediated regulation of genes in A. apis.

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.

Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous singlestranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.

【目的】 蜜蜂肠道是食物消化和营养吸收的主要部位，同时也是抵御病原侵染的的重要场所。本研究旨在对意大利蜜蜂Apis mellifera ligustica（简称意蜂）幼虫肠道的microRNAs（miRNAs）及其靶基因进行深入分析，进而解析miRNAs在幼虫肠道发育和生长过程中的作用。【方法】 利用small RNA-seq（sRNA-seq）技术对意蜂4、5和6日龄幼虫肠道进行测序，通过相关生物信息学软件对意蜂幼虫肠道的miRNAs进行预测及分析。利用TargetFinder软件预测miRNAs的靶基因，然后将靶基因通过BLAST软件进行GO和KEGG数据库的功能注释。利用Cytoscape软件构建miRNAs与其靶向结合的mRNAs的调控网络。【结果】 意蜂幼虫肠道样品的测序共获得96 329 456条有效标签序列（Clean tags），预测出560个miRNAs，包括45个novel miRNAs。上述miRNAs的长度主要分布在17-27 nt之间，且不同长度的miRNAs的首位碱基偏向性具有明显差异。表达量聚类分析结果显示，331个miRNAs为4、5和6日龄幼虫肠道所共有且表达量较为稳定，随着发育时间延长，特有miRNAs的表达水平呈总体增加的趋势。利用TargetFinder软件共预测出16 479个意蜂幼虫肠道的靶基因，其中有8 132和3 361个可分别注释到GO和KEGG数据库，进一步分析发现有224个靶基因注释在Wnt信号通路，分别有2个和27个靶基因与保幼激素和蜕皮激素的调控密切相关。【结论】 本研究在全基因组水平对意蜂幼虫肠道的miRNAs进行预测和分析，研究结果不仅丰富了意蜂的miRNAs信息，也为深入研究意蜂幼虫肠道的生长发育机理打下了初步基础。

【目的】蜜蜂球囊菌（Ascosphaera apis，简称球囊菌）是一种能够侵染中华蜜蜂（Apis cerana cerana，简称中蜂）幼虫的致死性真菌病原。微小RNA（microRNA，miRNA）可通过在转录后水平靶向抑制或降解mRNA而参与宿主与病原互作过程。本研究旨在对球囊菌胁迫的中蜂6日龄幼虫肠道的差异表达miRNA（DEmiRNA）及其靶基因进行深入分析，进而揭示DEmiRNA在蜜蜂响应球囊菌胁迫应答过程中的作用。【方法】利用Illumina MiSeq平台对正常及球囊菌胁迫的中蜂6日龄幼虫肠道（AcCK和AcT）进行测序，通过相关生物信息学软件预测DEmiRNA及其靶基因。通过Blast将靶基因注释到GO和KEGG数据库。利用Cytoscape软件构建DEmiRNA与其靶mRNAs的调控网络。通过Stem-loop RT-PCR和qPCR验证测序数据的可靠性。【结果】本研究共预测出537个miRNA，其长度分布介于16 ~35 nt之间，且不同长度的miRNA首位碱基偏向性差异明显。通过Stem-loop RT-PCR证实了10个novel miRNA的表达。AcCK vs AcT比较组共有54个DEmiRNA，包含31个上调和23个下调miRNA，可分别靶向结合6170 和8199个靶基因。GO分类结果显示上调和下调miRNA的靶基因分别涉及47和47个条目，富集基因数最多的皆为结合、细胞进程和催化活性。KEGG代谢通路（pathway）富集分析结果表明上调和下调miRNA的靶基因分别富集在134和126条pathway，富集基因数最多的均为内吞作用和内质网中的蛋白质加工。调控网络分析结果表明，DEmiRNA及其靶mRNAs形成十分复杂的调控关系；31个DEmiRNA可靶向结合51个与泛素介导的蛋白水解相关的mRNAs，18个DEmiRNA可靶向结合14个与Jak-STAT信号通路相关的mRNAs；miR-1277-x、miR-26-x、miR-27-y、miR-30-x、miR-6052-x等16个miRNA共同参与了上述两条免疫通路的调控。最后，随机挑选3个DEmiRNA进行qPCR验证，结果证明了测序数据的可靠性。【结论】本研究提供了中蜂幼虫肠道在球囊菌胁迫后期的miRNA的表达谱和差异表达信息，揭示了球囊菌与宿主之间在miRNA组学水平存在复杂的互作。miR-6052-x和miR-1277-x作为调控网络的核心可能通过影响细胞凋亡参与宿主的免疫防御，miR-26-x和miR-30-x可能通过调控Jak-STAT信号通路参与宿主的胁迫应答。本研究筛选出的关键DEmiRNA有望作为治疗白垩病的分子靶标。