Background/Objectives: Apis cerana development is described as comprising four stages: embryo, larva, pupa, and adult. There are significant differences between workers and drones in terms of physiological functions and social roles, and the formation of the organ primordia occurs during the embryonic stage. Therefore, the objective of this study is to investigate the differential expression of and alternative splicing of genes in worker and drone embryos and to explain their unique developmental patterns. Methods: Long-read sequencing (PacBio Iso-Seq) and short-read sequencing (Illumina RNA-Seq) were used to investigate worker and drone embryo gene expression differences in A. cerana across five developmental points (12, 24, 36, 48, and 60 h). Results: The study identified 59,254 common isoforms, with 5744 and 5106 isoforms specific to worker and drone embryos, respectively. Additionally, a new transcript of the csd gene was identified. The number of differentially expressed genes (3391) and differential splicing events (470 genes) peaked at the 24-h embryonic stage. Differential splicing events of csd, dsx, and Y-y were observed in the worker and drone embryos. Conclusions: The gene expression results indicated that the 24-h embryonic point is a critical period for the expression of genes related to developmental and behavioral differences between workers and drones. The findings provide a theoretical basis for future research on the developmental differences between workers and drones.

Embryonic development in honeybees is a critical stage that shapes the formation of organs and structures in adult bees. Although there has been substantial progress in transcriptome studies on honeybee embryogenesis, time-series transcriptomic and alternative splicing data in the embryonic development of the Asian honeybee (Apis cerana) remain limited. In this study, we conducted an in-depth analysis of RNA-seq data from public databases to examine the transcriptomic profiles at three key developmental stages of A. cerana embryos (24 h, 48 h, and 72 h), uncovering the dynamic changes in gene expression and alternative splicing across these stages. Results showed that the number of differentially expressed genes and alternative splicing events peaked at 24 and 48 h and then gradually decreased. Time-series transcriptomic analysis further identified key physiological and biochemical processes at these stages, reflecting a progression from foundational metabolism and cellular structure construction in the early stages to cell differentiation and organogenesis in the middle stage, and finally to functional structure refinement and behavioral trait formation in the later stage. Notably, our study highlighted the central role of alternative splicing and gene expression in driving key physiological and morphological changes during embryogenesis. We identified multiple key genes, including DMRT family genes, the Maelstrom (Mael) gene, and highly GO-enriched genes such as Dll, CaMKII, and Cnn. These genes not only play essential roles in structural formation but also support neurodevelopment and the emergence of complex behavioral patterns in adult bees. Gene expression and splicing patterns at different developmental stages provide new insights, revealing the early foundational mechanisms underlying limb development, behavior and memory, sensory organ development, and neural plasticity in honeybees.

The mitochondrial genome provides crucial information for understanding the evolution and phylogeny of various Apis cerana populations. Apis cerana -Southern China is a unique ecological type of Asian bee, A. cerana , primarily found in the coastal mountains of South China. Here, we used PacBio-HiFi sequencing to obtain the complete mitochondrial genome of A. cerana -Southern China and infer the phylogenetic relationships between A. cerana -Southern China and other A. cerana ecotypes. The mitochondrial genome of A. cerana -Southern China contains 16,137 bp and includes 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and an AT-rich region. The overall base composition is as follows: A (42.41%), C (9.59%), G (6.18%), and T (41.82%). The combined percentage of A and T (84.23%) is significantly higher than that of G and C. Among the 37 genes, 23 were located on the majority strand, while the remaining 14 were located on the minority strand. The phylogenetic tree based on the 13 PCGs showed that the genetic distance between A. cerana -Southern China, A. cerana -Central China, and A. cerana -Aba was closer. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in A. cerana -Southern China.

The honeybee Apis cerana plays a crucial role as a pollinator, contributing significantly to biodiversity and supporting ecological processes

Background/Objectives: The larval stage plays a pivotal role in determining caste and sex in Apis mellifera. This study integrates RNA-seq, WGCNA, and alternative splicing analyses to explore gene expression differences among 2-day-old worker, drone, and queen larvae. Methods: RNA-seq was conducted on 2-day-old larvae from all three castes. Differential expression, WGCNA, and alternative splicing patterns were investigated. A deep learning TCN model was trained using WGCNA-derived modules and demonstrated high classification accuracy. Results: The TCN model highlighted a top-10 gene set, including PDHB, Fibroin3, and LOC724161. Significant caste- and sex-specific splicing events were detected in Tk, Csd, and Fem, with AF events being most prevalent. Splicing differences between sexes exceeded those observed among castes. Conclusions: The 2-day-old larval stage is crucial for both caste and sex differentiation in honeybees. This study identifies key genes and splicing events, offering new insights into the molecular mechanisms underlying caste formation and sex determination.