Long non-coding RNAs (lncRNAs) are a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins, and lncRNAs have been proven to play pivotal roles in a wide range of biological processes in animals and plants. However, knowledge of expression patterns and potential roles of honeybee lncRNA response to Nosema ceranae infection is completely unknown. Here, we performed whole transcriptome strand-specific RNA sequencing of normal midguts of Apis mellifera ligustica workers (Am7CK, Am10CK) and N. ceranae-inoculated midguts (Am7T, Am10T), followed by comprehensive analyses using bioinformatic and molecular approaches. A total of 6353 A. m. ligustica lncRNAs were identified, including 4749 conserved lncRNAs and 1604 novel lncRNAs. 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 revealed here offer not only a rich genetic resource for further investigation of the functional roles of lncRNAs involved in the A. m. ligustica response to N. ceranae infection, but also a novel insight into understanding the host-pathogen interaction during honeybee microsporidiosis.

MiRNAs, as a kind of key regulators in gene expression, play vital roles in numerous life activities from cellular proliferation and differentiation to development and immunity. However, little is known about the regulatory manner of miRNAs in the development of Asian honey bee (Apis cerana) guts. Here, on basis of our previously gained high-quality transcriptome data, transcriptome-wide identification of miRNAs in the larval guts of Apis cerana cerana was conducted, followed by investigation of the miRNAs’ differential expression profile during the gut development. In addition to the regulatory network, the potential function of differentially expressed miRNAs (DEmiRNAs) was further analyzed. In total, 330, 351, and 321 miRNAs were identified in the 4-, 5-, and 6-day-old larval guts, respectively; among these, 257 miRNAs were shared, while 38, 51, and 36 ones were specifically expressed. Sequences of six miRNAs were confirmed by stem-loop RT-PCR and Sanger sequencing. Additionally, in the “Ac4 vs. Ac5” comparison group, there were seven up-regulated and eight down-regulated miRNAs; these DEmiRNAs could target 5041 mRNAs, involving a series of GO terms and KEGG pathways associated with growth and development, such as cellular process, cell part, Wnt, and Hippo. Comparatively, four up-regulated and six down-regulated miRNAs detected in the “Ac5 vs. Ac6” comparison group and the targets were associated with diverse development-related terms and pathways, including cell, organelle, Notch and Wnt. Intriguingly, it was noticed that miR-6001-y presented a continuous up-regulation trend across the developmental process of larval guts, implying that miR-6001-y may be a potential essential modulator in the development process of larval guts. Further investigation indicated that 43 targets in the “Ac4 vs. Ac5” comparison group and 31 targets in the “Ac5 vs. Ac6” comparison group were engaged in several crucial development-associated signaling pathways such as Wnt, Hippo, and Notch. Ultimately, the expression trends of five randomly selected DEmiRNAs were verified using RT-qPCR. These results demonstrated that dynamic expression and structural alteration of miRNAs were accompanied by the development of A. c. cerana larval guts, and DEmiRNAs were likely to participate in the modulation of growth as well as development of larval guts by affecting several critical pathways via regulation of the expression of target genes. Our data offer a basis for elucidating the developmental mechanism underlying Asian honey bee larval guts.

Nosema ceranae is a widespread fungal parasite for honey bees, causing bee nosemosis. Based on deep sequencing and bioinformatics, identification of circular RNAs (circRNAs) in Apis cerana workers’ midguts and circRNA-regulated immune response of host to N. ceranae invasion were conducted in this current work, followed by molecular verification of back-splicing sites and expression trends of circRNAs. Here, 10185 and 7405 circRNAs were identified in the midguts of workers at 7 days (AcT1) and 10 days (AcT2) post inoculation days post-inoculation with N. ceranae. PCR amplification result verified the back-splicing sites within three specific circRNAs (novel_circ_005123, novel_circ_007177, and novel_circ_015140) expressed in N. ceranae-inoculated midgut. In combination with transcriptome data from corresponding un-inoculated midguts (AcCK1 and AcCK2), 2266 circRNAs were found to be shared by the aforementioned four groups, whereas the numbers of specific ones were 2618, 1917, 5691, and 3723 respectively. Further, 83 52) differentially expressed circRNAs (DEcircRNAs) were identified in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group. Source genes of DEcircRNAs in workers’ midgut at seven dpi were involved in two cellular immune-related pathways such as endocytosis and ubiquitin mediated proteolysis. Additionally, competing endogenous RNA (ceRNA) network analysis showed that 23 13) DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group could target 18 14) miRNAs and further link to 1111 (1093) mRNAs. These target mRNAs were annotated to six cellular immunity pathways including endocytosis, lysosome, phagosome, ubiquitin mediated proteolysis, metabolism of xenobiotics by cytochrome P450, and insect hormone biosynthesis. Moreover, 284 164) internal ribosome entry site and 54 26) ORFs were identified from DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group; additionally, ORFs in DEcircRNAs in midgut at seven dpi with N. ceranae were associated with several cellular immune pathways including endocytosis and ubiquitin-mediated proteolysis. Ultimately, RT-qPCR results showed that the expression trends of six DEcircRNAs were consistent with those in transcriptome data. These results demonstrated that N. ceranae altered the expression pattern of circRNAs in A. c. cerana workers’ midguts, and DEcircRNAs were likely to regulate host cellular and humoral immune response to microsporidian infection. Our findings lay a foundation for clarifying the mechanism underlying host immune response to N. ceranae infection and provide a new insight into interaction between Asian honey bee and microsporidian.

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. mellifera. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (p < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (p < 0.001), while that in the inhibitor-miR-34 group was significantly downregulated (p < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibitor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5-, and 6-day-old larval guts were significantly downregulated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. mellifera larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.