Novel Insights into the circRNA-Modulated Developmental Mechanism of Western Honey Bee Larval Guts

SIMPLE SUMMARY: Current understanding of the regulatory part of circular RNAs (circRNAs) in the honey bee gut development is very limited. Here, 1728 circRNAs were detected in the larval guts of Apis mellifera workers. Seven among the most highly expressed 10 circRNAs were common among the 4-, 5-, and 6-day-old larval guts (Am4, Am5, and Am6 groups). Overall, 43 and 73 differentially expressed circRNAs (DEcircRNAs) were detected in the Am4 vs. Am5 and Am5 vs. Am6 comparison groups, respectively. These DEcircRNA parental genes were associated with an array of functional terms and pathways relevant to growth and development such as biological regulation and metabolic process. Novel_circ_000838 in the Am4 vs. Am5 comparison group potentially targeted ame-miR-6000a-3p, further targeting 518 mRNAs involved in 31 functional terms and 38 pathways, including five developmental signaling pathways and five immune pathways. The back-splicing sites and expression trends of six circRNAs were verified using molecular approaches. These results provide novel insights into the circRNA-modulated developmental mechanism of A. mellifera worker larval guts. ABSTRACT: Circular RNAs (circRNAs) are a class of novel non-coding RNAs (ncRNAs) that play essential roles in the development and growth of vertebrates through multiple manners. However, the mechanism by which circRNAs modulate the honey bee gut development is currently poorly understood. Utilizing the transcriptome data we obtained earlier, the highly expressed circRNAs in the Apis mellifera worker 4-, 5-, and 6-day-old larval guts were analyzed, which was followed by an in-depth investigation of the expression pattern of circRNAs during the process of larval guts development and the potential regulatory roles of differentially expressed circRNAs (DEcircRNAs). In total, 1728 expressed circRNAs were detected in the A. mellifera larval guts. Among the most highly expressed 10 circRNAs, seven (novel_circ_000069, novel_circ_000027, novel_circ_000438, etc.) were shared by the 4-, 5-, and 6-day-old larval guts. In addition, 21 (46) up-regulated and 22 (27) down-regulated circRNAs were, respectively, screened in the Am4 vs. Am5 (Am5 vs. Am6) comparison groups. Additionally, nine DEcircRNAs, such as novel_circ_000340, novel_circ_000758 and novel_circ_001116, were shared by these two comparison groups. These DEcircRNAs were predicted to be transcribed from 14 and 29 parental genes; these were respectively annotated to 15 and 22 GO terms such as biological regulation and catalytic activity as well as 16 and 21 KEGG pathways such as dorsoventral axis formation and apoptosis. Moreover, a complicated competing endogenous RNA (ceRNA) network was observed; novel_circ_000838 in the Am4 vs. Am5 comparison group potentially targeted ame-miR-6000a-3p, further targeting 518 mRNAs engaged in several developmental signaling pathways (e.g., TGF-beta, hedgehog, and wnt signaling pathway) and immune pathways (e.g., phagosome, lysosome, and MAPK signaling pathway). The results demonstrated that the novel_circ_000838-ame-miR-6000a-3p axis may plays a critical regulatory part in the larval gut development and immunity. Furthermore, back-splicing sites of six randomly selected DEcircRNAs were amplified and verified by PCR; an RT-qPCR assay of these six DEcircRNAs confirmed the reliability of the used high-throughput sequencing data. Our findings provide a novel insight into the honey bee gut development and pave a way for illustration of the circRNA-modulated developmental mechanisms underlying the A. mellifera worker larval guts.