Unveiling the circRNA-Mediated Immune Responses of Western Honey Bee Larvae to Ascosphaera apis Invasion.

Western honey bee (Apis mellifera), a eusocial insect with a superior economic and ecological value, is widely used in the beekeeping industry throughout the world. As a new class of non-coding RNAs (ncRNAs), circular RNAs (circRNAs) participate in the modulation of considerable biological processes, such as the immune response via diverse manners. Here, the identification, characteristic investigation, and molecular verification of circRNAs in the Apis mellifera ligustica larval guts were conducted, and the expression pattern of larval circRNAs during the Ascosphaera apis infection was analyzed, followed by the exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 2083 circRNAs in the larval guts of A. m. ligustcia were identified, with a length distribution ranging from 106 nt to 92,798 nt. Among these, exonic circRNAs were the most abundant type and LG1 was the most distributed chromosome. Additionally, 25, 14, and 30 up-regulated circRNAs as well as 26, 25, and 62 down-regulated ones were identified in the A. apis-inoculated 4-, 5-, and 6-day-old larval guts in comparison with the corresponding un-inoculated larval guts. These DEcircRNAs were predicted to target 35, 70, and 129 source genes, which were relative to 12, 23, and 20 GO terms as well as 11, 10, and 27 KEGG pathways, including 5 cellular and humoral immune pathways containing apoptosis, autophagy, endocytosis, MAPK, Toll, and Imd signaling pathways. Furthermore, complex competing endogenous RNA (ceRNA) regulatory networks were detected to be formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The Target DEmRNAs were engaged in 24, 20, and 25 functional terms as well as 62, 80, and 159 pathways, including several vital immune defense-associated pathways, namely the lysosome, endocytosis, phagosome, autophagy, apoptosis, MAPK, Jak-STAT, Toll, and Imd signaling pathways. Finally, back-splicing sites within 15 circRNAs and the difference in the 9 DEcircRNAs' expression between un-inoculated and A. apis-inoculated larval guts were confirmed utilizing molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions, but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. m. ligustica larvae against A. apis invasion.

Comprehensive investigation and regulatory function of lncRNAs engaged in western honey bee larval immune response to Ascosphaera apis invasion.

Ascosphaera apis is a fungal pathogen that exclusively infects bee larvae, causing chalkbrood disease, which results in severe damage for beekeeping industry. Long non-coding RNAs (lncRNAs) are versatile regulators in various biological processes such as immune defense and host-pathogen interaction. However, expression pattern and regulatory role of lncRNAs involved in immune response of bee host to A. apis invasion is still very limited. Here, the gut tissues of Apis mellifera ligustica 4-, 5-, and 6-day-old larvae inoculated by A. apis spores (AmT1, AmT2, and AmT3 groups) and corresponding un-inoculated larval guts (AmCK1, AmCK2, and AmCK3 groups) were prepared and subjected to deep sequencing, followed by identification of lncRNAs, analysis of differentially expressed lncRNAs (DElncRNAs), and investigation of competing endogenous RNA (ceRNA) network. In total, 3,746 A. m. ligustica lncRNAs were identified, including 78 sense lncRNAs, 891 antisense lncRNAs, 1,893 intergenic lncRNAs, 346 bidirectional lncRNAs, and 210 intronic lncRNAs. In the 4-, 5-, and 6- comparison groups, 357, 236, and 505 DElncRNAs were discovered. Additionally, 217, 129, and 272 DElncRNAs were respectively predicted to regulate neighboring genes via cis-acting manner, and these targets were associated with a series of GO terms and KEGG pathways of great importance, such as response to stimulus and Jak-STAT signaling pathway. Moreover, 197, 95, and 356 DElncRNAs were observed to target 10, eight, and 21 DEmiRNAs and further target 147, 79, and 315 DEmRNAs, forming complex regulatory networks. Further investigation suggested that these targets were engaged in several key cellular and humoral immune pathways, such as phagosome and MAPK signaling pathway. Ultimately, the expression trends of nine randomly selected DElncRNAs were verified by RT-qPCR, confirming the authenticity and reliability of our transcriptome data. Findings in this current work not only provide candidate DElncRNAs for functional study, but also lay a foundation for unclosing the mechanism underlying DElncRNA-regulated larval immune responses to A. apis invasion.

Deciphering the CircRNA-Regulated Response of Western Honey Bee (Apis mellifera) Workers to Microsporidian Invasion.

Vairimorpha ceranae is a widespread fungal parasite of adult honey bees that leads to a serious disease called nosemosis. Circular RNAs (circRNAs) are newly discovered non-coding RNAs (ncRNAs) that regulate biological processes such as immune defense and development. Here, 8199 and 8711 circRNAs were predicted from the midguts of Apis mellifera ligustica workers at 7 d (Am7T) and 10 d (Am10T) after inoculation (dpi) with V. ceranae spores. In combination with transcriptome data from corresponding uninoculated midguts (Am7CK and Am10CK), 4464 circRNAs were found to be shared by these four groups. Additionally, 16 circRNAs were highly conserved among A. m. ligustica, Apis cerana cerana, and Homo sapiens. In the Am7CK vs. Am7T (Am10CK vs. Am10T) comparison group, 168 (306) differentially expressed circRNAs (DEcircRNAs) were identified. RT-qPCR results showed that the expression trend of eight DEcircRNAs was consistent with that in the transcriptome datasets. The source genes of DEcircRNAs in Am7CK vs. Am7T (Am10CK vs. Am10T) were engaged in 27 (35) GO functional terms, including 1 (1) immunity-associated terms. Moreover, the aforementioned source genes were involved in three cellular immune-related pathways. Moreover, 86 (178) DEcircRNAs in workers' midguts at 7 (10) dpi could interact with 75 (103) miRNAs, further targeting 215 (305) mRNAs. These targets were associated with cellular renewal, cellular structure, carbohydrate and energy metabolism, and cellular and humoral immunity. Findings in the present study unraveled the mechanism underlying circRNA-mediated immune responses of western honey bee workers to V. ceranae invasion, but also provided new insights into host-microsporidian interaction during nosemosis.

Comparative Transcriptome Investigation of Nosema ceranae Infecting Eastern Honey Bee Workers.

Apis cerana is the original host for Nosema ceranae, a widespread fungal parasite resulting in honey bee nosemosis, which leads to severe losses to the apiculture industry throughout the world. However, knowledge of N. ceranae infecting eastern honey bees is extremely limited. Currently, the mechanism underlying N. ceranae infection is still largely unknown. Based on our previously gained high-quality transcriptome datasets derived from N. ceranae spores (NcCK group), N. ceranae infecting Apis cerana cerana workers at seven days post inoculation (dpi) and 10 dpi (NcT1 and NcT2 groups), comparative transcriptomic investigation was conducted in this work, with a focus on virulence factor-associated differentially expressed genes (DEGs). Microscopic observation showed that the midguts of A. c. cerana workers were effectively infected after inoculation with clean spores of N. ceranae. In total, 1411, 604, and 38 DEGs were identified from NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. Venn analysis showed that 10 upregulated genes and nine downregulated ones were shared by the aforementioned comparison groups. The GO category indicated that these DEGs were involved in a series of functional terms relevant to biological process, cellular component, and molecular function such as metabolic process, cell part, and catalytic activity. Additionally, KEGG pathway analysis suggested that the DEGs were engaged in an array of pathways of great importance such as metabolic pathway, glycolysis, and the biosynthesis of secondary metabolites. Furthermore, expression clustering analysis demonstrated that the majority of genes encoding virulence factors such as ricin B lectins and polar tube proteins displayed apparent upregulation, whereas a few virulence factor-associated genes such as hexokinase gene and 6-phosphofructokinase gene presented downregulation during the fungal infection. Finally, the expression trend of 14 DEGs was confirmed by RT-qPCR, validating the reliability of our transcriptome datasets. These results together demonstrated that an overall alteration of the transcriptome of N. ceranae occurred during the infection of A. c. cerana workers, and most of the virulence factor-related genes were induced to activation to promote the fungal invasion. Our findings not only lay a foundation for clarifying the molecular mechanism underlying N. ceranae infection of eastern honey bee workers and microsporidian-host interaction.

First Identification and Investigation of piRNAs in the Larval Gut of the Asian Honeybee, Apis cerana.

Piwi-interacting RNAs (piRNAs), a class of small non-coding RNAs (ncRNAs), play pivotal roles in maintaining the genomic stability and modulating biological processes such as growth and development via the regulation of gene expression. However, the piRNAs in the Asian honeybee (Apis cerana) are still largely unknown at present. In this current work, on the basis of previously gained high-quality small RNA-seq datasets, piRNAs in the larval gut of Apis cerana cerana, the nominated species of A. cerana, were identified for the first time, followed by an in-depth investigation of the regulatory roles of differentially expressed piRNAs (DEpiRNAs) in the developmental process of the A. c. cerana. Here, a total of 621 piRNAs were identified in A. c. cerana larval guts, among which 499 piRNAs were shared by 4-(Ac4 group), 5-(Ac5 group), and 6-day-old (Ac6 group) larval guts, while the numbers of unique ones equaled 79, 37, and 11, respectively. The piRNAs in each group ranged from 24 nucleotides (nt) to 33 nt in length, and the first base of the piRNAs had a cytosine (C) bias. Additionally, five up-regulated and five down-regulated piRNAs were identified in the Ac4 vs. Ac5 comparison group, nine of which could target 9011 mRNAs; these targets were involved in 41 GO terms and 137 pathways. Comparatively, 22 up-regulated piRNAs were detected in the Ac5 vs. Ac6 comparison group, 21 of which could target 28,969 mRNAs; these targets were engaged in 46 functional terms and 164 pathways. The results suggested an overall alteration of the expression pattern of piRNAs during the developmental process of A. c. cerana larvae. The regulatory network analysis showed that piR-ace-748815 and piR-ace-512574 in the Ac4 vs. Ac5 comparison group as well as piR-ace-716466 and piR-ace-828146 in the Ac5 vs. Ac6 comparison group were linked to the highest number of targets. Further investigation indicated that targets of DEpiRNAs in the abovementioned two comparison groups could be annotated to several growth and development-associated pathways, such as the Jak/STAT, TGF-ß, and Wnt signaling pathways, indicating the involvement of DEpiRNAs in modulating larval gut development via these crucial pathways. Moreover, the expression trends of six randomly selected DEpiRNAs were verified using a combination of stem-loop RT-PCR and RT-qPCR. These results not only provide a novel insight into the development of the A. c. cerana larval gut, but also lay a foundation for uncovering the epigenetic mechanism underlying larval gut development.