Background: Nutrition and cell size play an important role in the determination of caste differentiation in queen and worker of honeybees (Apis mellifera), whereas the haploid genome dominates the differentiation of drones. However, the effects of female developmental environment on the development of males remain unclear. In this study, young drone larvae were transferred into worker cells (WCs) or remained in drone cells (DCs) to rear drones. The drone larvae were also grafted into queen cells (QCs) for 48 h and then transplanted into drone cells until emerging. Morphological indexes and reproductive organs of these three types of newly emerged drones were measured. Newly emerged drones and third instar drone larvae from WCs, DCs and QCs were sequenced by RNA sequencing (RNA-Seq). Results: The amount of food remaining in cells of the QC and WC groups was significantly different to that in the DC group at the early larval stage. Morphological results showed that newly emerged DC drones had bigger body sizes and more well-developed reproductive tissues than WC and QC drones, whereas the reproductive tissues of QC drones were larger than those of WC drones. Additionally, whole body gene expression results showed a clear difference among three groups. At larval stage there were 889, 1761 and 1927 significantly differentially expressed genes (DEGs) in WC/DC, QC/DC and WC/QC comparisons, respectively. The number of DEGs decreased in adult drones of these three comparisons [678 (WC/DC), 338 (QC/DC) and 518 (WC/QC)]. A high number of DEGs were involved in sex differentiation, growth, olfaction, vision, mammalian target of rapamycin (mTOR), Wnt signaling pathways, and other processes. Conclusions: This study demonstrated that the developmental environment of honeybee females can delay male development, which may serve as a model for understanding the regulation of sex differentiation and male development in social insects by environmental factors.

Honey bees are important species for the study of epigenetics. Female honey bee larvae with the same genotype can develop into phenotypically distinct organisms (sterile workers and fertile queens) depending on conditions such as diet. Previous studies have shown that DNA methylation and histone modification can establish distinct gene expression patterns, leading to caste differentiation. It is unclear whether the histone methylation modification H3K4me1 can also impact caste differentiation. In this study, we analyzed genome-wide H3K4me1 modifications in both queen and worker larvae and found that H3K4me1 marks are more abundant in worker larvae than in queen larvae at both the second and fourth instars, and many genes associated with caste differentiation are differentially methylated. Notably, caste-specific H3K4me1 in promoter regions can direct worker development. Thus, our results suggest that H3K4me1 modification may act as an important regulatory factor in the establishment and maintenance of caste-specific transcriptional programs in honey bees; however, the potential influence of other epigenetic modifications cannot be excluded.

Honey bees, rather than rear queens with eggs and larvae from worker cells, prefer to rear new queens with eggs form queen cells, if available. This may be a result of long-term evolutionary process for honey bee colonies. However, the exact mechanism of this phenomenon is unclear. In this study, queens were reared with eggs from queen cells (F1-QE), eggs from worker cells (F1-WE), and two-day-old larvae from worker cells (F1-2L). Physiological indexes and the expression of the development-related genes ((Hexamerin (Hex110, Hex70b), Transferrin (Trf), and Vitellogenin (Vg)) of reared F1 generation queens were measured and compared. Furthermore, F2 generation queens were reared with one-day-old larvae from F1 queens, and the weight and ovariole count of reared F2 generation daughter queens were examined. Meanwhile, the expression of the development- and reproduction-related genes (Hex110, Hex70b, Trf, Vg, and Juvenile Hormone (Jh)) and immune detoxication-related genes (Hymenoptaecin, Abeacin, and CytP450) of reared F2 queens were further explored. We found that the F1-QE queens had the highest physiological indexes and higher Hex110 and Trf expression levels, while no significant difference was found in the expression of Hex70b and Vg among the three groups of F1 queens. In addition, the reared queens of F2-QE had the highest quality, with the highest development, reproduction, immune-detoxication genes’ expression levels. Our results revealed that the quality of reared offspring queens from high-quality mother queens was also high. These findings inform methods for rearing high-quality queens and highlight that a high-quality queen is essential for offspring colony growth and survival.