Here, the expression profiles and differentially expressed miRNAs (DEmiRNAs) in the midguts of Apis cerana cerana workers at 7 d and 10 d post-inoculation (dpi) with N. ceranae were investigated via small RNA sequencing and bioinformatics. Five hundred and twenty nine (529) known miRNAs and 25 novel miRNAs were identified in this study, and the expression of 16 predicted miRNAs was confirmed by Stem-loop RT-PCR. A total of 14 DEmiRNAs were detected in the midgut at 7 dpi, including eight up-regulated and six down-regulated miRNAs, while 12 DEmiRNAs were observed in the midgut at 10 dpi, including nine up-regulated and three downregulated ones. Additionally, five DEmiRNAs were shared, while nine and seven DEmiRNAs were specifically expressed in midguts at 7 dpi and 10 dpi. Gene ontology analysis suggested some DEmiRNAs and corresponding target mRNAs were involved in various functions including immune system processes and response to stimulus. KEGG pathway analysis shed light on the potential functions of some DEmiRNAs in regulating target mRNAs engaged in material and energy metabolisms, cellular immunity and the humoral immune system. Further investigation demonstrated a complex regulation network between DEmiRNAs and their target mRNAs, with miR-598-y, miR-252-y, miR-92-x and miR-3654-y at the center. Our results can facilitate future exploration of the regulatory roles of miRNAs in host responses to N. ceranae, and provide potential candidates for further investigation of the molecular mechanisms underlying eastern honeybeemicrosporidian interactions.

【目的】利用RNA seq技术对中华蜜蜂Apis cerana cerana（简称中蜂）幼虫肠道参考转录组进行de novo组装，并进行功能及代谢通路注释，进而利用该转录组数据进行中蜂幼虫的SSR分子标记鉴定。【方法】实验室条件下饲养中蜂幼虫，将纯化的球囊菌孢子饲喂3日龄幼虫，剖取4、5和6日龄幼虫肠道，液氮速冻。将健康幼虫肠道与感染蜜蜂球囊菌Ascosphaera apis（简称球囊菌）的幼虫肠道同时进行Illumina测序。通过对raw reads的过滤得到clean reads，利用Trinity软件组装得到unigenes。通过BLASTx（E-value＜10−5）比对NCBI Nr、Swiss-Prot、KOG和KEGG数据库，对unigenes进行功能和代谢通路注释。利用MISA软件对所有unigenes进行SSR搜索，并利用Primer Premier 5软件设计特异性SSR引物，通过常规PCR对来源于北京、辽宁兴城和四川成都的中蜂幼虫肠道样本进行SSR位点鉴定。【结果】中蜂幼虫肠道的RNA seq共得到35 670 000条reads，de novo组装得到43 557个unigenes，平均长度为898 nt。共有18 225个unigenes可被注释到上述公共蛋白数据库，单独注释到NCBI Nr、Swiss-Prot、KOG和KEGG数据库的unigenes数分别为3 899、443、37和10个。KOG注释结果显示，11 442条unigenes分布于25个基因家族，其中注释到RNA加工和修饰家族的基因数最多，达1 249个。9 679个unigenes的GO分类结果显示，在生物学进程分类中，注释到细胞进程的基因最多，达4 201个，在分子功能和细胞组分类中，注释到结合与细胞的基因数最多，分别为4 935和2900个。4 517个unigenes可注释到KEGG数据库中的216个代谢通路，注释到核糖体的基因数最多，达385个。利用MISA软件，可在7 763个unigenes搜索到13 448个SSR位点，随机选取20对SSR引物对国内3个不同来源的中蜂幼虫肠道样本的SSR位点进行扩增，有6对引物可鉴定出SSR分子标记。【结论】本研究成功组装并注释了中蜂幼虫肠道参考转录组，可为中蜂及其幼虫的分子生物学及组学研究提供重要的参考信息，也可用于补充、丰富和检验东方蜜蜂的参考基因组，基于此转录组数据开发出6个中蜂的SSR分子标记，可应用于中蜂的基因图谱构建、基因多样性分析、基因定位等研究，也说明利用转录组数据开发非模式生物SSRs的方法可行。

Chalkbrood is a fungal disease of honey bees and leads to serious apicultural loss worldwide. Ascosphaera apis, the pathogen of chalkbrood, has been found in Apis mellifera, Apis cerana, Xylocopa californica arizonensis larvae as well as adult bumble bees. Here, the fungal pathogen of Apis cerana cerana drone mummies was isolated and identified using morphological and molecular methods. Morphological observation indicated that the sizes of the fruiting bodies, spore balls and ascospores of the fungus isolated from A. c. cerana drone mummies were similar to those of A. apis. Phylogenetic analysis suggested that the fungus is identical to A. apis. Furthermore, the results of a cross infection assay demonstrated that the isolated fungus is capable of infecting eastern and western honey bee larvae and results in chalkbrood. These results confirmed that the isolated fungal pathogen is A. apis. This is the first documentation of morphological and molecular identification of A. apis in A. c. cerana. Our results not only provide novel insight to better understand the pathology of A. apis, but also lay a solid foundation for further investigations of host responses and pathogen-host interactions during chalkbrood of eastern honey bee larvae.

东方蜜蜂微孢子虫Nosema ceranae是专性寄生蜜蜂中肠上皮细胞的单细胞真菌，对意大利蜜蜂(意蜂)具有较强的侵染性。本研究利用 ＲNA-seq 技术对正常 10 d ( Apis mellifera ligustica control group，AmCK)和N. ceranae胁迫10 d的意蜂工蜂中肠(Apis mellifera ligustica treatment group，AmT)进行测序，共得到160 847 237 100 条原始读段， 过滤后得到 1 066 955 298 条有效读段。主成分分析结果显示 AmCK 与 AmT 测序样品的组内重复性较好， 组间的基因表达模式差异明显。GO 分类结果显示， AmCK与AmT的前100 位高表达基因( HEGs)分别分布于32和33个GO terms， 基因富集数最多的均为代谢进程。KEGG 代谢通路(pathway)富集分析结果显示，AmCK的前100位HEGs富集在21个pathways，基因富集数最多的是内吞作用、信号通路和嘌呤代谢; AmT的前100 位 HEGs 富集在26个pathways，基因富集数最多的是内吞作用、ＲNA 转运和蛋白质的内质网加工。前100位HEGs的Venn分析结果显示AmCK与AmT的共有HEGs为87个，特有HEGs分别均为13个。研究结果揭示了N. ceranae胁迫意蜂工蜂中肠过程中的基因表达谱信息，也为解析N. ceranae致病的分子机理提供了有益信息和线索。

Nosema ceranae is a unicellular fungal parasite of honey bees and causes huge losses for apiculture. Until present, no study on N . ceranae long non-coding RNAs (lncRNAs) was documented. Here, we sequenced purified spores of N . ceranae using strand-specific library construction and high-throughput RNA sequencing technologies. In total, 83 novel lncRNAs were predicted from N . ceranae spore samples, including lncRNAs, long intergenic non-coding RNAs (lincRNAs), and sense lncRNAs. Moreover, these lncRNAs share similar characteristics with those identified in mammals and plants, such as shorter length and fewer exon number and transcript isoforms than protein-coding genes. Finally, the expression of 12 lncRNAs was confirmed with RT-PCR, confirming their true existence. To our knowledge, this is the first evidence of lncRNAs produced by a microsporidia species, offering novel insights into basic biology such as regulation of gene expression of this widespread taxonomic group.