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

Abstract

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-β, 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.

Diverse Regulatory Manners and Potential Roles of lncRNAs in the Developmental Process of Asian Honey Bee (Apis cerana) Larval Guts

“…Our previous work suggested that miRNAs and piRNAs are differentially expressed and potentially participate in the developmental processes of the larval guts of A. c. cerana workers. Collectively, these results demonstrate that various types of ncRNAs, including lncRNAs, miRNAs, and piRNAs, are likely to participate in the modulation of larval gut development through single regulation or co-regulation via cross-talk, as verified in previous studies [20,29]. A further analysis showed that the expression levels of several lncRNAs were continuously elevated with an increase in the developmental time, which is suggestive of their pivotal roles in the developmental process, thus deserving further functional investigations.…”

“…In honey bees, previous studies have mainly focused on intestinal microorganisms [26][27][28], and few advancements in the developmental mechanisms of bee guts have been gained until now. In recent years, on the basis of deep sequencing and bioinformatics, we systematically analyzed the differential expression profiles and putative regulatory roles of piRNAs and miRNAs in the developmental process of A. cerana worker larval guts [29,30]. In this current work, the expression pattern of lncRNAs during the developmental process of the guts of A. c. cerana worker larvae was surveyed based on our previously gained high-quality transcriptome data, followed by an in-depth investigation of the regulatory manners and potential roles of DElncRNAs in the larval guts.…”

Unraveling the modulatory manner and function of circRNAs in the Asian honey bee larval guts

“…Findings from other groups have shown that not only circRNA but also other ncRNAs such as miRNAs, piRNAs, and lncRNAs were involved in the regulation of the development of insects ( Maeda et al, 2018 ; Betting et al, 2021 ; Quesnelle et al, 2023 ). Previously, our team observed the differential expression of miRNAs, piRNAs, and lncRNAs in the developmental process of the A. cerana worker larval guts, and uncovered that DEmiRNAs, DEpiRNAs, and DElncRNAs were putative modulators in the larval gut development ( Fan et al, 2023a ; b ; Long et al, 2022 ). In summary, these results suggested that various types of ncRNAs were likely to regulate the development of A. cerana worker larval guts through diverse manners such as ceRNA networks as both circRNAs and lncRNAs could interact with miRNAs ( Tay et al, 2014 ; Zhang et al, 2019 ).…”

Expression Profile, Regulatory Network, and Putative Role of microRNAs in the Developmental Process of Asian Honey Bee Larval Guts

“…However, few advancements in the developmental mechanism of the bee guts have been gained until now. Recently, we resolved the expression patterns, regulatory networks, and potential functions of piRNAs, another kind of small ncRNAs, in the larval guts of both A. mellifera and A. cerana [ 32 , 33 ]. However, little is known about the miRNA-regulated developmental mechanism underlying the bee gut at present.…”