A Global Survey of the Full-Length Transcriptome of Apis mellifera by Single-Molecule Long-Read Sequencing.

As important pollinators, honey bees play a crucial role in both maintaining the ecological balance and providing products for humans. Although several versions of the western honey bee genome have already been published, its transcriptome information still needs to be refined. In this study, PacBio single-molecule sequencing technology was used to sequence the full-length transcriptome of mixed samples from many developmental time points and tissues of A. mellifera queens, workers and drones. A total of 116,535 transcripts corresponding to 30,045 genes were obtained. Of these, 92,477 transcripts were annotated. Compared to the annotated genes and transcripts on the reference genome, 18,915 gene loci and 96,176 transcripts were newly identified. From these transcripts, 136,554 alternative splicing (AS) events, 23,376 alternative polyadenylation (APA) sites and 21,813 lncRNAs were detected. In addition, based on the full-length transcripts, we identified many differentially expressed transcripts (DETs) between queen, worker and drone. Our results provide a complete set of reference transcripts for A. mellifera that dramatically expand our understanding of the complexity and diversity of the honey bee transcriptome.

Expression Profile of microRNAs during Development of the Hypopharyngeal Gland in Honey Bee, Apis mellifera.

The hypopharyngeal gland is an important organ for honey bees to secrete royal jelly, and its secretory activity varies with the age of workers. However, by now, the regulation mechanism of hypopharyngeal gland development is still unclear. Here, the expression profiles of miRNAs in the hypopharyngeal gland of newly emerged workers, nurses, and foragers were investigated via small RNA sequencing. From these three stages, 81 known miRNAs and 135 novel miRNAs have been identified. A total of 85 miRNAs showed expression differences between different development stages, and their target genes were predicted to range from 1 to more than 10. Many of the differentially expressed miRNAs and target genes are related to growth and development or apoptosis. Moreover, dual-luciferase-reporter assays verified that novel-miR-11 directly targets the 3'-untranslated regions of LOC410685 (inactive tyrosine-protein kinase 7) and LOC725318 (uncharacterized protein). These results suggested that miRNAs were widely involved in the developmental regulation of the hypopharyngeal gland in honey bees.

Revealing Changes in Ovarian and Hemolymphatic Metabolites Using Widely Targeted Metabolomics between Newly Emerged and Laying Queens of Honeybee (Apis mellifera).

The queen bee is a central and pivotal figure within the colony, serving as the sole fertile female responsible for its reproduction. The queen possesses an open circulatory system, with her ovaries immersed in hemolymph. A continuous and intricate transportation and interchange of substances exist between the ovaries and hemolymph of queen bees. To determine the characteristic metabolites in the hemolymph and ovary, as well as understand how their rapid metabolism contributes to the process of egg-laying by queens, we reared Apis mellifera queens from three different age groups: newly emerged queen (NEQ), newly laying queen (NLQ), and old laying queen (OLQ). Using widely targeted metabolomics, our study revealed that the laying queen (NLQ and OLQ) exhibited faster fatty acid metabolism, up-regulated expression of antioxidants, and significant depletion of amino acids compared to the NEQ. This study revealed that the levels of carnitine and antioxidants (GSH, 2-O-α-D-glucopyranosyl-L-ascorbic acid, L-ascorbic acid 2-phosphate, etc.) in the NLQ and OLQ were significantly higher compared to NEQ. However, most of the differentially expressed amino acids, such as L-tryptophan, L-tyrosine, L-aspartic acid, etc., detected in NLQ and OLQ were down-regulated compared to the NEQ. Following egg-laying, pathways in the queens change significantly, e.g., Tryptophan metabolism, Tyrosine metabolism, cAMP signaling pathway, etc. Our results suggest that carnitine and antioxidants work together to maintain the redox balance of the queen. Additionally, various amino acids are responsible for maintaining the queen's egg production.

The Honey Bee Colony's Criterion for Candidate Selection: "Ongoing" or "One-Shot"?

In the honey bee, the queen's death severely threatens the survival of the colony. In an emergency, new queens are reared from young worker larvae, where nepotism is thought to influence the choice of queen candidates by the workers. This article simulates the emergency queen-rearing process in a colony under natural conditions and records the results of colony selection (without nepotism). In queenless colonies, worker larvae aged three days or younger were preferred for queen rearing, and 1-day-old larvae were the first to be selected for the queen-cell cups. In the capping stage, the number of capped queen cells selected from the 1-day-old larvae was much higher than the 3-day-old larvae. On the first day, the number of emerging queens reared from 1-day-old larvae was significantly higher than the queens reared from 2-day-old and 3-day-old larvae. However, there was no significant difference in the birth weights of queens reared from 1-day-old, 2-day-old, or 3-day-old larvae. When the newly emerged queens were introduced into the original queenless colony, 1-day-old larval queens triggered more worker followers than 2-day-old larval queens. The expression of ovarian development-related genes (vg, hex110, and Jh) was higher in queens reared from 1-day-old larvae than those reared from 2-day-old and 3-day-old larvae, indicating that the quality of the queens reared from 1-day-old larvae is superior. This study shows that in the absence of nepotism, the colony selection of queen candidates at the larval stage, capping stage, and emerging stage is not final, but is gradually optimized to maximize colony development through a "quality control" process.

Transcriptome changes of Apis mellifera female embryos with fem gene knockout by CRISPR/Cas9.

The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees.