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.

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.

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.

Long non-coding RNAs (lncRNAs) have been proven to play pivotal roles in a wide range of biological processes in animals and plants. Here, we performed whole transcriptome strand-specific RNA sequencing of normal midguts of Apis mellifera ligustica workers (Am7CK, Am10CK) and Nosema ceranae-inoculated midguts (Am7T, Am10T), and 4749 conserved lncRNAs and 1604 novel lncRNAs were identified. These lncRNAs had minimal sequence similarities with other known lncRNAs in other species; however, their structural features were similar to counterparts in mammals and plants, including shorter exon and intron length, lower exon number, and lower expression level, compared with protein-coding transcripts. Further, 111 and 146 N. ceranae-responsive lncRNAs were identified from midguts at 7-days post-inoculation (dpi) and 10 dpi compared with control midguts. Twelve differentially expressed lncRNAs (DElncRNAs) were shared by Am7CK vs. Am7T and Am10CK vs. Am10T comparison groups, while the numbers of unique DElncRNAs were 99 and 134, respectively. Functional annotation and pathway analysis showed that the DElncRNAs may regulate the expression of neighboring genes by acting in cis and trans fashion. Moreover, we discovered 27 lncRNAs harboring eight known miRNA precursors and 513 lncRNAs harboring 2257 novel miRNA precursors. Additionally, hundreds of DElncRNAs and their target miRNAs were found to form complex competitive endogenous RNA (ceRNA) networks, suggesting that these DElncRNAs may act as miRNA sponges. Furthermore, DElncRNA-miRNA-mRNA networks were constructed and investigated, the results demonstrated that a portion of the DElncRNAs were likely to participate in regulating the host material and energy metabolism as well as cellular and humoral immune host responses to N. ceranae invasion. Our findings offer a rich genetic resource for further investigation of the functional roles of lncRNAs involved in the A. m. ligustica response to N. ceranae infection.

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.