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.

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 down-regulated 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 honeybee-microsporidian interactions.

Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, is a common disease among cage-farmed large yellow croaker (Larimichthys crocea) in China. However, comprehensive investigations of the molecular defensive mechanisms used by L. crocea in response to P. plecoglossicida infection remain relatively rare. Here, we constructed transcriptomes of the L. crocea spleen at 12 h and 24 h after P. plecoglossicida challenge. We identified 518 novel miRNAs and 823 known miRNAs in the spleen of L. crocea. Between the challenge and control groups, 32 differentially expressed miRNAs (DEmiRNAs), predicted to target 356 genes, and 1152 differentially expressed mRNAs (DEmRNAs) were identified at 12 h post-infection, while 33 DEmiRNAs, predicted to target 278 genes, and 1067 DEmRNAs were identified at 24 h post-infection. Gene ontology (GO) analysis showed that 146 and 126 GO terms were significantly enriched in the target genes at 12 h and 24 h, respectively. Twenty-eight and four immune-associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the target genes at 12 h and 24 h, respectively. Three immune-associated pathways were among those most enriched in the target genes: Toll-like receptor signaling, endocytosis, and C-type lectin receptor signaling. Network analysis identified 47 DEmRNA-DEmiRNA pairs. In particular, the immune-related genes TLR5S and PIGR were targeted by the miRNAs lcr-miR-7132c and dre-miR-183-5p, respectively. Dual-luciferase assays verified that lcr-miR-7132c downregulated TLR5S, suggesting that this miRNA may participate in regulating the immune response of L. crocea to P. plecoglossicida infection through the TLR5S-mediated signaling pathway. Our results help to clarify the miRNA-mediated immune response of L. crocea to P. plecoglossicida infection.