1. Introduction
As a representative social insect with crucial ecological, economic and scientific value, the western honey bee (Apis mellifera) is widely reared and applied in the apicultural industry in considerable countries around the world [1]. In addition, A. mellifera has been applied as a research model for development, social behavior, epigenomics, gene regulation and host–pathogen/parasite interaction [2]. As early as 2006, the first version of A. mellifera genome assembly (Amel_4.0) was published by the Honey Bee Genome Sequencing Consortium (2006). Recently, based on PacBio, 10x Chromium, BioNano, and Hi-C [3], Wallberg et al. reconstructed the A. mellifera reference genome (Amel_Hav3.1), which lays a solid foundation for dissecting the biology of A. mellifera at the molecular level.
Circular RNAs (circRNAs), a type of single-stranded, covalently closed, and endogenous non-coding RNAs (ncRNAs), are capable of producing a closed-loop structure connecting the 5′ and 3′ ends by a non-canonical “reverse splicing” process [4]. This unique structure makes circRNAs more stable than linear RNAs and highly resistant to degradation mediated by the exonuclease RNase R [5,6]. Hereafter, following the revolution and development of RNA sequencing (RNA-seq) technology and related bioinformatics, more and more circRNAs have been discovered in various animals, plants, and microorganisms, such as Drosophila [7], Bombyx mori [8], Homo sapiens [9], Mus musculus [10], Oryza sativa L. [11], Triticum aestivum L. [12], Ascosphaera apis [13], and Nosema ceranae [14]. CircRNAs have been demonstrated to exert diverse and vital functions in numerous life activities such as gene regulation and development through various manners, including interaction with RNA polymerase II to facilitate the transcription of host genes as well as action modulation of the expression and activity of target miRNAs [15,16,17,18].
The gut tissue of insects is a major tissue for the digestion of foods, absorption of nutrition, and immune defense against various pathogens or parasites [19,20,21]. Both miRNAs and long non-coding RNAs (lncRNAs) have been demonstrated to participate in regulating the growth and development of the insect gut. For example, Foronda et al. [22] reported that miR-305 was involved in adjusting the balance between differentiation and self-renewal of Drosophila gut stem cells by regulating Notch and insulin signaling pathways, enabling adaptive homeostasis in the gut to respond to changing environmental conditions. Wang et al. [23] discovered that lncR26319 was able to modulate Endophilin A (EndoA) through the competitive absorption of miR-2834, thereby increasing the endocytic activity in the vitellogenin (Vg) uptake, which gave rise to the normal progression of Bombyx mori egg production. However, little advancements in the regulatory function of circRNAs in the developmental process of insect gut have been achieved.
Accumulating studies have shown that circRNAs were vital regulators in many aspects of honey bees, such as ovary activation and oviposition [24], brain aging and division of labor [25], immune response [26], and host–parasite interaction [27]. Based on deep sequencing and transcriptomic investigation, our group discovered that circRNAs were likely to regulate the responses of both A. mellifera and Apis cerana to infections by two widespread fungal pathogens including A. apis and N. ceranae [27,28]. By analyzing the expression profile and modulatory part of circRNAs in the midgut of Apis cerana workers, Chen et al. [29] discovered that circRNAs were potentially engaged in modulating the development of Apis cerana cerana workers’ midgut tissues through diverse ways like the ceRNA network as well as the regulation of neighboring gene transcription. The developmental stage of honey bee larvae lasts for six days. The gut tissue of adult honey bees can roughly be divided into the foregut, midgut, and hindgut; however, it is hard to precisely distinguish different sections of the honey bee larval gut [30,31]. At present, the modulatory manner and role of circRNAs in the A. mellifera larval guts is still completely unknown.
Previously, the worker larval gut samples of A. m. ligustica, a subspecies of A. mellifera widely used in the beekeeping industry, were prepared followed by the deep sequencing of cDNA libraries [32]. Our data could provide not only a new perspective into the honey bee gut development but also a foundation for elucidating the developmental mechanism of the larval guts.
4. Discussion
Currently, following investigating expression levels, we detected that among the top 10 highly expressed circRNAs, 7 (novel_circ_000069, novel_circ_000027, novel_circ_000438, etc.) were shared by the A. m. ligustica worker 4-, 5-, and 6-day-old larval guts (Table 1), which is indicative of the great importance of these seven common circRNAs during the process of larval gut development, thus deserving further investigation. Additionally, 43 and 73 DEcircRNAs were, respectively, identified in the Am4 vs. Am5 and Am5 vs. Am6 comparison groups (Figure 2; see also Tables S5 and S6), which suggested that the development of larval guts was accompanied by the dynamic change in the overall expression profile of circRNAs. In other animals such as Aedes albopictus (Diptera: Culicidae), Bombyx mori, and Drosophila, the change in expression pattern of circRNAs was also detected in the developmental process. For example, Liu et al. [47] found that circRNA can act as hub genes, manipulate chitin metabolism, and further promote the growth and development of Aedes albopictus (Diptera: Culicidae). Wang et al. [48] reported that the expression level of circEgg in the Bombyx mori midgut dynamically changed during the developmental process and circEgg was potentially involved in regulating the homeostasis of midgut tissue. These findings reflected that circRNAs are possibly involved in the regulation of the development of honey bee and other animals. Interestingly, two up-regulated circRNAs (novel_circ_000758, novel_circ_001116) were observed to be shared by the above-mentioned two comparison groups, suggesting the potential part of them in the development of larval gut tissue. These DEcircRNAs are believed to be candidates for further functional investigation using our recently established RNAi-based methods [49].
Several lines of evidence have demonstrated that circRNAs exert regulatory functions in diverse aspects of vertebrates such as growth, development, metabolism, and immunity through cis-acting effect [50,51,52,53]. For instance, Weigelt et al. [51] previously detected the continuous up-regulation of the circRNA transcribed from the sulfateless gene (circSfl) in the brain and muscle of insulin mutant flies, and the circSfl overexpression prolonged the host lifespan. Zhang et al. [52] discovered that the interference of circRNA2030 was capable of suppressing the expression of its parental gene phospholipid-transporting ATPase (PTA), and it can enhance the infectivity of RBSDV for Laodelphax striatellus (Fallen) midgut tissues; thus, the authors speculated that circRNA2030 was likely to control RBSDV infection via PTA regulation. Here, the parental genes of DEcircRNAs were relative to several vital functional terms and pathways associated with growth, development, metabolism, and immunity, such as biological regulation, catalytic activity, and multicellular organismal process (Figure 3A,B; see also Tables S7 and S8). The results indicated that corresponding DEcircRNAs potentially affected the aforementioned functional terms and pathways of great importance during the gut’s developmental process. Additionally, it is noticed that novel_circ_000758 was differentially expressed in both Am4 vs. Am5 and Am5 vs. Am6 comparison groups, and the parental gene (ncbi_413021) of novel_circ_000758 was involved in the metabolic process, catalytic activity, and extracellular matrix (Figure 3A,B; see also Tables S9 and S10), indicating the key role of novel_circ_000758 in the gut development. Thus, it is worth further study in the near future.
Papilins are secreted extracellular matrix proteins, homologous among various species ranging from nematodes to humans [54]. Papillin is an extracellular stromal glycoprotein and is associated with the thin stroma layer during gastrula formation, the stroma associated with phagocytic blood cells, the basement membrane, and the space-filling stroma during drosophila development [55,56]. In this study, the differential expression of novel_circ_000758 was detected in the Am4 vs. Am5 and Am5 vs. Am6 comparison groups, and the parental gene (GeneBank accession number: LOC413021) of novel_circ_000758 was annotated as the papilin protein. It is speculated that novel_circ_000758 played an essential role in regulating the gut development of the larval gut by regulating the transcription of genes encoding papilin.
Apolipophorins are carrier proteins by binding lipids in animals, and they mediate the transfer of lipids between tissues [57]. Also, apolipophorins are engaged in stress response and lipid transport in insects [58] such as mosquito [59] and Locusta migratoria [57]. In this current work, the parental gene (GeneBank: LOC408961) of novel_circ_001116, a DEcircRNA in the Am4 vs. Am5 and Am5 vs. Am6 comparison groups, was annotated as apolipophorins protein, which suggested that novel_circ_001116 may be a modulator in the lipid transport and immune response of A. mellifera worker larval gut through affecting the transcription of apolipophorin-encoded genes. Further work is needed to unclose the functions and molecular mechanisms of novel_circ_000758 and novel_circ_001116.
It has been suggested that circRNAs are crucial regulators in immune defense of insects against pathogens or parasites [60]. Hu et al. [61] investigated the circRNA expression in the gut tissue of Bombyx mori following BmNPV challenge, and the results showed that circRNAs potentially regulate the genes annotated to ubiquitin, apoptosis, and endocytosis signaling pathways by the ceRNA axis. The antiviral defense system of honey bees has been suggested to include hemocyte-mediated mechanisms of cellular immune (e.g., endocytosis, melanization and phagosome), and conserved signaling pathways (e.g., MAPK, and FoxO signaling pathways) [62]. Here, we detected that two parental genes (ncbi_551176, ncbi_100577393) of novel_circ_000838 and novel_circ_000861 in the Am4 vs. Am5 comparison group were associated with phagosome and endocytosis, which are two insect cellular immune pathways of great importance; one parental gene (ncbi_727312) of novel_circ_001933 in the Am5 vs. Am6 comparison group was enriched in endocytosis (Figure 3C; see also Tables S9 and S10). For insects like the honey bee, the gut is a critical immune organ that fights against pathogenic microorganisms through cellular and humoral immune pathways [63,64]. Here, in the Am4 vs. Am5 comparison group, one (ncbi_100577393) and two (ncbi_725827 and ncbi_100577393) parental genes of novel_circ_000861, novel_circ_000400, and novel_circ_000861 were observed to be, respectively, enriched in the MAPK and FoxO signaling pathways (Figure 3C; see also Tables S9 and S10). Based on the findings mentioned above, corresponding DEcircRNAs were speculated to participate in the adjustment of immune responses in the process of the gut’s development.
Increasing studies have demonstrated that circRNAs containing miRNA response elements (MREs) could act as “sponges” to bind to target miRNAs and indirectly affect the downstream gene expression [65,66]. Gao et al. [67] documented that circRNA-407 was capable of promoting the expression of its target gene Foxl via targeting aal-miR-9a-5p and eventually regulated the Aedes albopictus ovarian development; the siRNA-mediated knockdown of circRNA-407 leads to a depression in follicle number and follicle size during the stage of ovarian development. In the Am4 vs. Am5 comparison group, novel_circ_000838 that significantly down-regulated was detected to putatively interact with ame-miR-6000a-3p, further targeting a subseries of mRNAs relevant to not only catalytic activity, the metabolic process, the immune system process and biological regulation but also three developmental signaling pathways (Wnt, TGF-beta, and Hedgehog) and two cellular immune pathways (phagosome and endocytosis) (Figure 4B; see also Table S13). The results indicated that miR-6000a-3p may be not only a pivotal regulator in diverse processes in the honey bee such as gut growth and development but also a hub to bridge other ncRNAs like circRNAs and genes. The functions of the majority of miRNAs in the honey bee larval guts, including ame-miR-6000a-3p, are currently unknown. Therefore, more efforts for the functional dissection of A. mellifera larval miRNAs should be undertaken. Recently, our group established the feeding-based method of functional study on miRNAs in the gut tissue of A. mellifera larvae [68], offering a platform for investigating the function of ame-miR-6000a-3p and continuous investigation of other miRNAs. Additionally, the technical platform was recently established for the functional dissection of bee larval circRNA by our research team [49]. Together, these platforms pave the way for exploring the functions as well as mechanisms of circRNAs, miRNAs, and ceRNA axis in the honey bee larvae.
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