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.

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.

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.

【目的】本研究利用small RNA-seq技术对球囊菌的纯培养进行测序，对球囊菌的microRNAs(miRNAs)进行预测、鉴定和分析，进而构建miRNAs-mRNAs的调控网络。【方法】利用Illumina Hiseq Xten平台对球囊菌菌丝与孢子进行测序，通过相关生物信息学软件对球囊菌的miRNAs进行预测和分析，通过茎环(Stem-loop)PCR对部分miRNAs进行鉴定，利用Cytoskype软件构建miRNAs-mRNAs的调控网络。【结果】本研究共获得48268696条clean reads，预测出118个球囊菌的miRNAs，它们的长度分布介于18–25 nt之间，不同长度的miRNA的首位碱基偏好性差异明显。Stem-loop PCR验证结果显示共有10个miRNAs能够扩增出符合预期的目的片段，说明多数miRNAs可能真实存在。共预测出6529个球囊菌miRNAs的靶基因，其中的5725个能够注释到Nr、Swissprot、KOG、GO和KEGG数据库。进一步分析结果显示有24个靶基因注释在MAPK信号通路。Cytoskype软件分析结果显示球囊菌的miRNAs与mRNAs之间存在复杂的调控网络，绝大多数的miRNAs处于调控网络的内部且同时结合多个mRNAs。【结论】本研究率先对球囊菌的miRNAs及miRNAs-mRNAs调控网络进行全面分析，研究结果丰富了对球囊菌miRNAs的认识，为其基础生物学信息提供了有益补充，也为阐明球囊菌致病的分子机理打下了一定基础。

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.