MiRNAs, as a kind of key regulators in gene expression, play vital roles in numerous life activities from cellular proliferation and differentiation to development and immunity. However, little is known about the regulatory manner of miRNAs in the development of Asian honey bee (Apis cerana) guts. Here, on basis of our previously gained high-quality transcriptome data, transcriptome-wide identification of miRNAs in the larval guts of Apis cerana cerana was conducted, followed by investigation of the miRNAs’ differential expression profile during the gut development. In addition to the regulatory network, the potential function of differentially expressed miRNAs (DEmiRNAs) was further analyzed. In total, 330, 351, and 321 miRNAs were identified in the 4-, 5-, and 6-day-old larval guts, respectively; among these, 257 miRNAs were shared, while 38, 51, and 36 ones were specifically expressed. Sequences of six miRNAs were confirmed by stem-loop RT-PCR and Sanger sequencing. Additionally, in the “Ac4 vs. Ac5” comparison group, there were seven up-regulated and eight down-regulated miRNAs; these DEmiRNAs could target 5041 mRNAs, involving a series of GO terms and KEGG pathways associated with growth and development, such as cellular process, cell part, Wnt, and Hippo. Comparatively, four up-regulated and six down-regulated miRNAs detected in the “Ac5 vs. Ac6” comparison group and the targets were associated with diverse development-related terms and pathways, including cell, organelle, Notch and Wnt. Intriguingly, it was noticed that miR-6001-y presented a continuous up-regulation trend across the developmental process of larval guts, implying that miR-6001-y may be a potential essential modulator in the development process of larval guts. Further investigation indicated that 43 targets in the “Ac4 vs. Ac5” comparison group and 31 targets in the “Ac5 vs. Ac6” comparison group were engaged in several crucial development-associated signaling pathways such as Wnt, Hippo, and Notch. Ultimately, the expression trends of five randomly selected DEmiRNAs were verified using RT-qPCR. These results demonstrated that dynamic expression and structural alteration of miRNAs were accompanied by the development of A. c. cerana larval guts, and DEmiRNAs were likely to participate in the modulation of growth as well as development of larval guts by affecting several critical pathways via regulation of the expression of target genes. Our data offer a basis for elucidating the developmental mechanism underlying Asian honey bee larval guts.

PiRNAs are a class of small non-coding RNAs that play essential roles in modulating gene expression and abundant biological processes. To decode the piRNA-regulated larval response of western honeybees (Apis mellifera) to Ascosphaera apis infection, the expression pattern of piRNAs in Apis mellifera ligustica larval guts after A. apis inoculation was analyzed based on previously obtained high-quality small RNA-seq datasets, followed by structural characterization, target prediction, regulatory network investigation, and functional dissection. Here, 504, 657, and 587 piRNAs were respectively identified in the 4-, 5-, and 6-day-old larval guts after inoculation with A. apis, with 411 ones shared. These piRNAs shared a similar length distribution and first base bias with mammal piRNAs. Additionally, 96, 103, and 143 DEpiRNAs were detected in the 4-, 5-, and 6-day-old comparison groups. Targets of the DEpiRNAs were engaged in diverse pathways such as the phosphatidylinositol signaling system, inositol phosphate metabolism, and Wnt signaling pathway. These targets were involved in three energy metabolism-related pathways, eight development-associated signaling pathways, and seven immune-relevant pathways such as the Jak-STAT signaling pathway. The expression trends of five randomly selected DEpiRNAs were verified using a combination of RT-PCR and RT-qPCR. The effective overexpression and knockdown of piR-ame-945760 in A. apis-infected larval guts were achieved by feeding a specific mimic and inhibitor. Furthermore, piR-ame-945760 negatively regulated the expression of two target immune mRNAs, SOCS5 and ARF1, in the larval gut during the A. apis infection. These findings indicated that the overall expression level of piRNAs was increased and the expression pattern of piRNAs in larval guts was altered due to the A. apis infection, DEpiRNAs were putative regulators in the A. apis-response of A. m. ligustica worker larvae. Our data provide not only a platform for the functional investigation of piRNAs in honeybees, especially in bee larvae, but also a foundation for illuminating the piRNA-involved mechanisms underlying the host response to the A. apis infection.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

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.

Safe overwintering is a challenging issue in rearing management that is inevitably faced by beekeepers in high-latitude regions. Under the combined influence of multiple factors, the overwintering loss rate of Western honey bees has risen continuously, and investigating the molecular mechanisms related to safe overwintering has become key. The Hunchun bee, an Apis mellifera ecotype in Jilin Province, China, exhibits strong overwintering ability during an overwintering period of more than five months. To investigate the molecular mechanisms of its cold resistance, we conducted a comparative transcriptomic analysis between the summer breeding period (July) and different overwintering intervals (November, December, January, and February), and then systematically identified key genes and signaling pathways related to cold resistance. The results showed that the highest number of differentially expressed genes (DEGs) was found between December and July. Compared with July, the upregulated genes in Hunchun bee in December were significantly enriched in several pathways, such as ion transport and neuroactive ligand–receptor interactions, and the downregulated genes were significantly enriched in pathways related to fatty acid metabolism, glutathione metabolism, and the peroxisome. Notably, a total of 378 shared DEGs were obtained from the four comparison groups, and several candidate cold-resistant gene families, such as AFPs, HSPs, C₂H₂-ZFPs, STKs, and LRRCs, were identified among the shared DEGs of the winter season. Additionally, 749 shared DEGs related to protein modification and metabolic process regulation were identified between the four successive overwintering intervals. Four shared genes, including sensory neuron membrane protein 1 (SNMP1), were revealed by pairwise comparison of the four intervals. The above results collectively indicate that the Hunchun bee attenuates winter-induced stress responses during the overwintering process by maintaining osmotic pressure balance, reducing fatty acid metabolism, increasing antioxidant capacity, and synthesizing cold-resistant macromolecular proteins. It was also found that chemical signal perception may serve a role in maintaining the stability of the overwintering bee colony. The key genes and pathways related to cold resistance identified in this study not only provide a basis for explaining the overwintering molecular mechanism for Apis mellifera of Hunchun bee but also offer key data to improve overwintering management strategies for Western honey bees.

The conservation and breeding of the western honey bee (Apis mellifera) is central dependent on accurate subspecies assignment, but the most commonly used methods are labor-intensive classical morphometrics and costly molecular assays. We developed an XGBoost-based classification framework using a compact set of routinely measurable characters. A curated dataset of labeled workers was measured under harmonized protocols; features were screened according to embedded importance, and model performance was assessed using five-fold cross-validation, outperforming standard machine learning baselines. The resulting model using only the top 10 characters—primarily forewing venation angles and abdominal plate metrics—achieved high performance (accuracy = 0.98; F1 = 0.99) and an area under the receiver operating characteristic curve (AUC) of 0.99 (95% CI = 0.995–0.999). SHAP analyses confirmed the discriminatory contributions of these features, while error inspection suggested that misclassifications were concentrated in morphologically overlapping lineages. The model’s performance supports its use as a rapid triage tool alongside genetic testing, providing a scalable and interpretable tool for researchers to create and deploy custom morphometric models, demonstrated here for A. mellifera but portable to other insect taxa.