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.

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.

Apis cerana is the original host for Nosema ceranae, a widespread fungal parasite resulting in honey bee nosemosis, which leads to severe losses to the apiculture industry throughout the world. However, knowledge of N. ceranae infecting eastern honey bees is extremely limited. Currently, the mechanism underlying N. ceranae infection is still largely unknown. Based on our previously gained high-quality transcriptome datasets derived from N. ceranae spores (NcCK group), N. ceranae infecting Apis cerana cerana workers at seven days post inoculation (dpi) and 10 dpi (NcT1 and NcT2 groups), comparative transcriptomic investigation was conducted in this work, with a focus on virulence factor-associated differentially expressed genes (DEGs). Microscopic observation showed that the midguts of A. c. cerana workers were effectively infected after inoculation with clean spores of N. ceranae. In total, 1411, 604, and 38 DEGs were identified from NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. Venn analysis showed that 10 upregulated genes and nine downregulated ones were shared by the aforementioned comparison groups. The GO category indicated that these DEGs were involved in a series of functional terms relevant to biological process, cellular component, and molecular function such as metabolic process, cell part, and catalytic activity. Additionally, KEGG pathway analysis suggested that the DEGs were engaged in an array of pathways of great importance such as metabolic pathway, glycolysis, and the biosynthesis of secondary metabolites. Furthermore, expression clustering analysis demonstrated that the majority of genes encoding virulence factors such as ricin B lectins and polar tube proteins displayed apparent upregulation, whereas a few virulence factor-associated genes such as hexokinase gene and 6-phosphofructokinase gene presented downregulation during the fungal infection. Finally, the expression trend of 14 DEGs was confirmed by RT-qPCR, validating the reliability of our transcriptome datasets. These results together demonstrated that an overall alteration of the transcriptome of N. ceranae occurred during the infection of A. c. cerana workers, and most of the virulence factor-related genes were induced to activation to promote the fungal invasion. Our findings not only lay a foundation for clarifying the molecular mechanism underlying N. ceranae infection of eastern honey bee workers and microsporidian–host interaction.

Royal jelly is a specific product secreted by honeybees, and has been sought after to maintain health because of its valuable bioactive substances, e.g., lipids and vitamins. The lipids in royal jelly come from the bee pollen consumed by honeybees, and different plant source of bee pollen affects the lipid composition of royal jelly. However, the effect of bee pollen consumption on the lipid composition of royal jelly remains unclear. Herein, we examined the influence of two factors on the lipid composition of royal jelly: first, two plant sources of bee pollen, i.e., Acer mono Maxim. (BP-Am) and Phellodendron amurense Rupr. (BP-Pa); secondly, different feeding times. Lipidomic analyses were conducted on the royal jelly produced by honeybees fed BP-Am or BP-Pa using ultra-high performance liquid chromatography (UPLC)-Q-Exactive Orbitrap mass spectrometry. The results showed that the phospholipid and fatty acid contents differed in royal jelly produced by honeybees fed BP-Am compared to those fed BP-Pa. There were also differences between timepoints, with many lipid compounds decreasing in abundance soon after single-pollen feeding began, slowly increasing over time, then decreasing again after 30 days of single-pollen feeding. The single bee pollen diet destroyed the nutritional balance of bee colonies and affected the development of hypopharyngeal and maxillary glands, resulting in differences in royal jelly quality. This study provides guidance for optimal selection of honeybee feed for the production of high-quality royal jelly.

The division of labor is a dominant characteristic of honeybees and is accompanied by behavioral specialization and cognitive enhancement. As the central nervous system to control the labor-specific behaviors of honeybee, the brain is richest in lipid in terms of both diversity and abundance. In this study, an in-depth LC-MS/MS-based lipidomic method was applied to systematically characterize the brain lipid compositions of worker bees with three labor stages: newly emerged bee (NEB), nurse bee (NB), and forager bee (FB). A total number of 337 lipid species that assigned to 20 lipid classes were analyzed. The association of the brain lipidomes with the division of labors was suggested by the results of both the unsupervised and supervised multivariate pattern recognition analysis. More than 68% of the identified lipid species were found to be significantly changed in at least one comparison between NEB, NB, and FB. A total of 81 lipid species were identified as the potential labor-featured molecules with VIP > 1 and p-adj < 0.05. The labor-featured lipids of FA(18:2), FA(18:3), FA(26:0), PC(18:0_18:3), PS(18:1_18:1), SM(d38:1), CoQ10, and CoQ9, as well as their interactions with 12 behavior-related genes, including AmEST-6, AmFABP, AmE75, AmDGAT2, AmLSD1, AmNPC1, AmABCA1, AmNMDAR1, AmHTT, AmNOS, etc., were revealed by the further IPA analysis. These findings demonstrate for the first time that the brain lipidomes of worker bees are associated with the stable differences in their labors, which help understand the function of brain lipids on the labor-dependent behaviors of honeybees.

The technology of long reads substantially improved the contingency of the genome assembly, particularly resolving contiguity of the repetitive regions. By integrating the interactive fragment using Hi-C, and the HiFi technique, a solid genome of the honeybee Apis mellifera carnica was assembled at the chromosomal level. A distinctive pattern of genes involved in social evolution was found by comparing it with social and solitary bees. A positive selection was identified in genes involved with cold tolerance, which likely underlies the adaptation of this European honeybee subspecies in the north hemisphere. The availability of this new high-quality genome will foster further studies and advances on genome variation during subspeciation, honeybee breeding and comparative genomics.