Honey bee is one of the most important pollinating insects in nature and plays an essential part in ecological balance and food security (Tan et al., 2022). Apis cerana is a major bee species widely reared in many parts of Asian countries, with a subseries of advantages like adaptation to diverse environments, ability of fast dispersal, ecological balance maintenance and crop pollination (Gallai et al., 2009). The gut of insects including honeybee is a major tissue responsible for food digestion, nutrient absorption, and immune defense (Holtof et al., 2019; Khan et al., 2023). However, the developmental mechanisms underlying the honeybee gut is currently still largely unknown.

Non-coding RNAs (ncRNAs), such as miRNA, lncRNA, and circular RNA (circRNA), represent a layer of regulators in various biological processes (Zhang et al., 2019). NcRNAs can be divided into housekeeping ncRNAs and regulatory ncRNAs, and the latter is usually considered to play a critical part in controlling gene expression at transcriptional or post-transcriptional level (Leng et al., 2022). As a new member of regulatory ncRNA family, circRNAs are generated by the back-splicing of pre-mRNAs (Li et al., 2018b). Since circRNAs have special covalently closed-loop structure that lacks 5′cap and 3′ployA tail, circRNAs are more resistant to RNase R enzyme digestion than linear RNAs and therefore regarded as ideal endogenous biomarker (Meng et al., 2017; Kristensen et al., 2019).

Abundant circRNAs have been discovered in animals, plants, and microorganisms, such as H. sapiens (Liu et al., 2019), Triticum aestivum (Xu et al., 2019) and Varroa destructor (Lin et al., 2021). Accumulating evidence have shown that circRNAs played vital roles in diverse processes, such as development, metabolism, and immunity (Liu et al., 2019; Zhou et al., 2020; Shen et al., 2024). Recent studies on human diseases suggested that circRNAs can serve as novel biomarkers, therapeutic agents, and drug targets (Xin et al., 2021; Kristensen et al., 2022). Compared to mammals like Homo sapiens, study regarding circRNAs in insects is lagging. Limited advancements have gained in a few model insects such as Drosophila (Weigelt et al., 2020), Aedes albopictus (Gao et al., 2023), and Bombyx mori (Wang et al., 2020). For instance, Gao et al. (2023) demonstrated that circRNA-407 acted as a sponge of aal-miR-9a-5p to promote the expression of the target gene Foxl and eventually modulated ovarian development. In recent years, the findings from other groups and our team suggested that circRNAs were potential regulators in western honey bee (Apis mellifera) ovary activation and oviposition (Chen et al., 2019a; Chen et al., 2023 X.), brain nerve apoptosis (Shan et al., 2023), task allocation (Thölken et al., 2019), and midgut growth and development (Guo et al., 2018). Chen X. et al. (2023) detected that overexpression of ame_circ_0002015 enhanced the number of eggs laid by A. mellifera queens, and further verified that ame_circ_0002015 was capable of sponging ame_miR-14-3p.

Current understanding of A. cerana circRNAs is even more scarce. Chen et al. (2020) previously identified 9,589 circRNAs in the midgut tissues of A. cerana workers and revealed that differentially expressed circRNAs (DEcircRNAs) between 8- and 11-day-old group were likely to participate in the developmental process of midgut through diverse manners such as competing endogenous RNA (ceRNA). By deciphering the circRNA responses of A. cerana to infections by Ascosphaera apis and N. ceranae, our group observed that the expression pattern of host circRNAs was altered by these two widespread fungal bee pathogens and DEcircRNAs were involved in host responses including immune response (Zhu et al., 2022; Guo et al., 2024). However, it is still unknown whether and how circRNAs regulate the growth and development of the larval guts of A. cerana. Therefore, it is necessary and meaningful to investigate the regulatory manners and functions of circRNAs in the development of A. cerana larval guts.

In the present study, based on our previously obtained high-quality transcriptome datasets, we investigated the differential expression profile of circRNAs during the developmental process of A. cerana worker larval gut, followed by dissection of the regulatory manner and role of DEcircRNAs. Additionally, we analyzed the DEcircRNA-engaged sub-networks associated with immune pathways as well as developmental signaling pathways. Furthermore, the binding relationships between key DEcircRNA and target DEmiRNA as well as DEmiRNA and target DEmRNA were verified by molecular method. Findings from this current work offer a novel insight into the epigenetic regulation of the development of A. cerana worker larval gut, provide candidate DEcircRNA/DEmiRNA/DEmRNA axis for elucidating the mechanism underlying gut development.

Increasing studies have demonstrated that circRNAs were essential players in the developmental regulation of insects including A. mellifera (Krishnamoorthy and Kadener, 2021; Liu et al., 2022). Here, we detected 224 and 158 DEcircRNAs in the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, respectively, including 42 shared ones (Figure 1). This indicated that the developmental process of A. cerana larval guts was accompanied with the differential expression of the overall circRNA profile, suggestive of the potential roles of these DEcircRNAs in the larval gut development. 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).

As yet, circRNAs have been verified to control the transcription of parental genes and further affect a series of biological processes like development and metabolism (Gao et al., 2023), immune response (Yin et al., 2021), and inhibiting cell proliferation (Li et al., 2018a). Li et al. (2018a) found that circITGA7 upregulated the transcription of its parental ITGA7 by suppressing RREB1 via the Ras pathway and inhibits the proliferation and metastasis of colorectal cancer (CRC) cells. Therefore, functional annotation and pathway analysis of corresponding parental genes have profound significance for understanding the role of circRNAs. For insects, the gut tissue is the major position for material metabolism (Zhang and Edgar, 2022). It’s found in this study that the parental genes of DEcircRNAs in the above-mentioned two comparison groups were engaged in 11 and seven metabolism-related pathways, respectively, such as glycerolipid metabolism, amino sugar and nucleotide sugar metabolism, and arginine biosynthesis (Figure 2E, see also Supplementary Figure S1). The results indicated that corresponding DEcircRNAs were potentially involved in modulating material metabolisms in the larval guts by regulating parental genes’ transcription.

The development of insect gut is a complex process modulated by different signaling pathways, such as Hippo, Wnt, and Hedgehog (Jacob and Lum, 2007; Shaw et al., 2010; Kuroda et al., 2012). Hippo signaling pathway plays a central part in controlling cell proliferation and fate as well as organ growth and regeneration (Ma et al., 2019). Here, novel_circ_001365 (log₂FC = −17.68, p = 0.01) in the Ac4 vs. Ac5 comparison group was detected to downregulated and putatively regulated the transcription of the parental gene (ncbi_107997224) enriched in the Hippo signaling pathway. Also, two DEcircRNAs in the Ac5 vs. Ac6 comparison group, including novel_circ_001373 (log₂FC = 3.18, p = 0.03) and novel_circ_002848 (log₂FC = −1.25, p = 0.0003), were respectively observed to modulate the transcription of two parental genes (ncbi_107995510 and ncbi_108002348) involved in the Hippo signaling pathway. The results showed that novel_circ_001365, novel_circ_001373, and novel_circ_002848 may participate in the larval gut development via regulating the Hippo signaling pathway. Studies have shown that the Hedgehog signaling pathway is closely associated with the development of various organs or tissues, such as gastrointestinal tract, endocrine gland, skeletal development, and wing (van den Brink, 2007; Cohen, 2010; Ehlen et al., 2006; Vervoort, 2000). In this work, novel_circ_000076 (log₂FC = −17.68, p = 0.01) and novel_circ_002942 (log₂FC = −17.57, p = 0.03) in the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison group, was putatively modulated the transcription of ncbi_108004420 and ncbi_108002826, respectively, two genes engaged in the Hedgehog signaling pathway. This implied that novel_circ_000076 and novel_circ_002942 may regulate the development of larval gut in a Hedgehog signaling pathway-depending manner. Wnt signaling pathway plays a regulatory role of great importance in the development of organs in conjunction with the Hippo, Notch, and TGF-β signaling pathways (Sun and Wang, 2003; Barry and Camargo, 2013). Here, novel_circ_003093 (log₂FC = −1.40, p = 5 × 10⁻¹¹) was downregulated in the Ac4 vs. Ac5 comparison group and potentially regulated the transcription of a Wnt signaling pathway-relevant parental gene (ncbi_108003317), while novel_circ_002848 and novel_circ_002851 (log₂FC = 1.49, p = 0.003) in the Ac5 vs. Ac6 comparison group respectively regulated the transcription of ncbi_108002348 and ncbi_108002373, two parental genes enriched in the Wnt signaling pathway, which suggested that these three DEcircRNAs were putative regulators in the gut development. Additionally, mTOR and AMPK are considered master regulators of cell metabolism and their activation is directly linked to the regulation of cellular metabolism, growth and survival (Garza-Lombó et al., 2018); similarly, mTOR controls Insulin signaling by regulating several downstream components (Yoon, 2017); TGF-β signaling pathway also plays an important role in cellular growth (Massagué and Sheppard, 2023). Here, novel_circ_000850 (log₂FC = 17.77, p = 0.01) in the Ac4 vs. Ac5 comparison group and novel_circ_001609 (log₂FC = 17.73, p = 0.01) in the Ac5 vs. Ac6 comparison group regulated the transcription of ncbi_107993192 and ncbi_107996586, respectively, involved in the AMPK signaling pathway. In the Ac4 vs. Ac5 comparison group, novel_circ_001175 (log₂FC = 1.05, p = 0.02) and novel_circ_002087 (log₂FC = −2.83, p = 0.0002) were respectively observed to modulate the transcription of ncbi_107994726 and ncbi_107998974, these two genes engaged in the Insulin signaling pathway. In the Ac5 vs. Ac6 comparison group, novel_circ_002848 (log₂FC = −1.25, p = 0.0003) could regulate the transcription of ncbi_108002348, which could enrich in TGF-β signaling pathway. The above results suggested that these DEcircRNAs play a role in the development of the A. cerana larval guts by regulating the AMPK Insulin, TGF-β and mTOR signaling pathways. Among them, we found that in the Ac4 vs. Ac5 comparison group, novel_circ_002977 (log₂FC = −3.19, p = 1 × 10⁻¹¹) and novel_circ_002978 (log₂FC = −2.04, p = 1 × 10⁻⁶) were both observed to modulate the transcription of ncbi_108002698, and the parental gene ncbi_108002698 could involve in the AMPK, mTOR, and Insulin signaling pathway. This implied that novel_circ_002977 and novel_circ_002978 may regulate the development of larval gut through the combined action of mTOR/AMPK signaling pathway and Insulin signaling pathway. This also provides an important idea for our future research, next, we may explore the role of novel_circ_002977 and novel_circ_002978 in the development of the A. cerana worker larval guts in more detail. Together, these results demonstrated that corresponding DEcircRNAs were likely to modulate the Hippo, Hedgehog, Wnt, mTOR, AMPK, Insulin, and TGF-β signaling pathways by regulating the transcription of parental genes, further affecting the developmental process of the A. cerana worker larval guts.

The insect gut is a critical immune organ responsible for detection and defense against invading pathogens or parasites (Ratcliffe, 1985; Vilmos and Kurucz, 1998). In the present study, the parental genes of DEcircRNAs in the Ac4 vs. Ac5 comparison group were associated with four cellular immune pathways (lysosome, endocytosis, phagosome, and melanogenesis) and four humoral immune pathways (MAPK signaling pathway, MAPK signaling pathway-fly, cAMP signaling pathway, and Ras signaling pathway) (Figure 2C), whereas the parental genes of DEcircRNAs in the Ac5 vs. Ac6 comparison group were relative to nine immune pathways such as apoptosis and JAK-STAT signaling pathway (Supplementary Figure S1). In addition, the parental genes of three DEcircRNAs (novel_circ_002852, novel_circ_002853, novel_circ_002687) shared by these two comparison groups were found to regulate the parental genes engaged cAMP signaling pathway, and MAPK signaling pathway. The results were indicative of the involvement of corresponding DEcircRNAs in modulating the cellular and humoral immune in the larval gut during the developmental process.

Accumulating evidence have shown that various types of ncRNAs, such as circRNAs and lncRNAs, are able to regulate diverse activities of insects like development and immunity through ceRNA networks using miRNAs as a bridge (Gao et al., 2023; Ren et al., 2023). CircRNAs, mRNAs, and any other RNAs that share common miRNA response elements (MREs) can competitively bind with miRNAs, acting as an RNA sponge to block and inhibit miRNAs from binding to their target sites (Tay et al., 2014). Studies found that ceRNA regulatory networks are widely present in the growth and development of various insects, such as A. albopictus (Liu et al., 2023), Tetranychus cinnabarinus (Feng et al., 2022), B. mori (Yin et al., 2021). Here, we noticed that 19 DEcircRNAs (novel_circ_002117, novel_circ_001037, and novel_circ_002747, etc.) in the Ac4 vs. Ac5 comparison group could simultaneously target four DEmiRNAs (ace-novel-m0032-5p, ace-miR-6001-y, ace-miR-6001-x, and ace-miR-980-y), further targeting 14 DEmRNAs (ncbi_107993074, ncbi_108001413, and ncbi_108003740, etc.) which engaged in Hippo, Wnt, Hedgehog, mTOR, AMPK, Insulin, and TGF-β signaling pathways (Figure 5A); equally, nine DEcircRNAs such as novel_circ_000090 and novel_circ_002624 in the Ac5 vs. Ac6 comparison group could bind with ace-miR-6001-y and than targeted 11 DEmRNAs which also involved in Hippo, Wnt, Hedgehog, etc. seven signaling pathways (Figure 5B). The above results indicated that circRNAs in A. cerana worker larval guts development by regulating these seven signaling pathways through the ceRNA regulatory networks. Moreover, it also suggested that circRNAs can regulate the same pathway in different regulatory ways (regulating the transcription of parental genes and ceRNA mechanism). Insect Notch signaling plays remarkably diverse roles in development to regulate cell fate determination, organ growth and tissue patterning (Chen Y. et al., 2023). And the Notch signaling plays essential roles during ovary development (Shcherbata et al., 2004), leg growth (Cordoba and Estella, 2020), and wing development (Blair, 2007) in Drosophila melanogaster. In this work, we noticed that 15 DEcircRNAs in the Ac4 vs. Ac5 comparison group targeted ace-miR-6001-y further targeting ncbi_107998104 which enriched in Notch signaling pathway. Unlike the way of regulating the transcription of parental genes, the Notch signaling pathway is only regulated by the ceRNA mechanism, so we speculated the Notch signaling pathway may be a special pathway that can be regulated by the ceRNA mechanism during the development of A. cerana worker larval guts. In addition, these target DEmRNAs were annotated to seven (eight) cellular immune pathways and five (eight) humoral immune pathways, including melanogenesis, lysosome, Jak-STAT signaling pathway, and cAMP signaling pathway (Figure 4). This confirmed that the corresponding DEcircRNAs were involved in the modulation of immunity during gut development in A. cerana larvae.

Collins et al. (2017) isolated and validated Bte-miR-6001-5p and Bte-miR-6001-3p, and found that Bte-miR-6001-5p and Bte-miR-6001-3p were more highly expressed in queen-than in worker-destined late-instar larvae in Bombus terrestris. Shan et al. (2023) indicated that ame-miR-6001-3p was a key factor in circRNA-mediated ceRNA regulatory networks in the A. mellifera brain tissue which exposed to fluvalinate. Furthermore, ame-miR-6001-5p was found to play an essential role in ecdysone secretion and caste differentiation in A. mellifera (Shi et al., 2015; Ashby et al., 2016). Recently, Fan et al. (2023b) analyzed the expression pattern, regulatory network, and putative role of miRNAs through the small RNA-seq data from A. cerana worker larvae, and found that ace-miR-6001-y was upregulated in both Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups and adjusted some great importance signaling pathways (Wnt, Hippo, and Jak-STAT signaling pathways, etc.). More recently, Fan et al. (2023a) found that 54 and 116 DElncRNAs in the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, respectively, acted as ceRNAs to competitively target ame-miR-6001-y and regulated the development of A. cerana worker larval guts. Here, 15 DEcircRNAs in the Ac4 vs. Ac5 comparison group, such as novel_circ_000382, novel_circ_001276, and novel_circ_002424, were observed to target ace-miR-6001-y, further targeting 173 DEmRNA, involving eight (Hippo, Wnt, and AMPK signaling pathway, etc.) and 11 (necroptosis, melanogenesis, and lysosome, etc.) pathways relevant to development and immunity, respectively (Figure 6B). Additionally, ace-miR-6001-y was observed to be targeted by nine DEcircRNAs (novel_circ_000090, novel_circ_001627, and novel_circ_002651, etc.) in the Ac5 vs. Ac6 comparison group and further targeted 177 DEmRNAs (Figure 6C). These targets were engaged in nine development-related pathways including Hippo signaling pathway and Hedgehog signaling pathway as well as 16 immune-related pathways including MAPK signaling pathway and apoptosis. These results together demonstrated that corresponding DEcircRNAs were likely to absorb ace-miR-6001-y and further affect the development and immunity of the A. cerana larval guts. In recent years, adaptors have been shown to be associated with the development of ventral and dorsal compartments of the wing imaginal disc in Drosophila (Williams et al., 1993; Fernández-Fúnez et al., 1998). Here, we randomly selected novel_circ_001627/ace-miR-6001-y/adaptors-like (ncbi_107995149) axis for dual-luciferase reporter assay. The results verified the authenticity of the bonding relationship (Figure 9) and indicated that novel_circ_001627 and ace-miR-6001-y were possibly involved in the developmental process of A. cerana. This laied the groundwork for a more in-depth exploration in the future.

To summarize, circRNAs were dynamically expressed in the development of A. cerana workers larval gut. And during the developmental process, DEcircRNAs could be involved in regulation through their parental genes and as ceRNAs to compete with mRNAs to bind the same miRNAs (Figure 10). Some essential pathways (Hedgehog, Wnt, and Hippo signaling pathway) and the DEcircRNA/ace-miR-6001-y/DEmRNA axis have been found to act during the development of A. cerana workers larval guts. In a word, all these results reveal the role of cricRNA regulation during development. Dual-luciferase reporter assay verified the novel_circ_001627/ace-miR-6001-y/adaptors-like axis’ target relationships and provides a basis for corresponding circRNAs research.