Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager). Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs. In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

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.

The mechanism of sex determination is common for all honeybee species (Apis spp.) by the complementary sex determi-nation (csd) gene. The csd gene has been studied in the Western honey bee (Apis mellifera L.), the Eastern honey bee (Apis cerana F.) and the giant honey bee (Apis dorsata F.), whereas no studies had been conducted on the high altitude Himalayan or black giant honey bee, Apis laboriosa Smith. In the present study, we cloned the genomic exon 6 to exon 9 region of the A. laboriosa csd gene, and identified 13 csd haplotypes. The data was analyzed and compared with the other aforementioned three honeybee species. The results showed that, as with the other three Apis species, region 3 of the csd gene contains an RS domain at the N terminal, a proline-rich domain at the C terminal, and a hypervariable region in the middle. A phylogenetic tree showed that the csd haplotypes from A. laboriosa fell into one clade with those from A. dorsata, and were separated from those from the other two species, A. mellifera and A. cerana. The network map also showed that the csd haplotypes from A. laboriosa and A. dorsata are well mixed among each other, and do not form two separate branches. Pairwise Fst analysis revealed that the value between A. laboriosa and A. dorsata was very low (0.098), confirming a close relationship to exist between them.

Honey bees are vital pollinators in agriculture and important model insects. To understand the genetic and molecular aspects in their development, a reverse transcription quantitative polymerase chain (RT-qPCR) is used to investigate the target genes. However, it is essential to use the appropriate reference genes as endogenous controls for accurate normalization of target genes. To identify stable reference genes in two honey bee species, [Apis mellifera (Am) and Apis cerana (Ac)], we evaluated eight candidate reference genes including, actin, atub, ef1α, gapdh, rpl13a, rpl32, rps18 and tif. Worker bees belonging to the two species were collected at each developmental day during the embryonic and postembryonic developmental stages. The tyrosine hydroxylase (th) gene was used as the target gene to validate the selected reference genes. Our results revealed that rpl13a was the most stable reference gene at all developmental stages of Am and Ac. In addition, gene combinations, including Amrpl13a & Amrps18 & Amactin, Amrpl13a & Amrpl32, Acrpl13a & Acrpl32, Acrpl13a & Acrpl32 & Acef1α followed by other combinations effectively normalized the expression of the target genes during the embryonic and postembryonic developmental stages of Am and Ac, respectively. Our findings provide a foundation for standardized RT-qPCR analysis to improve the accuracy of genes normalization during the different developmental stages of honey bees.