Female developmental environment delays development of male honeybee (Apis mellifera)

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 hrs 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 3-day 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 relatively larger than those of WC drones. Additionally, gene expression results showed a clear difference among three groups. At the 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. 

Conclusion

This study demonstrated that the developmental environment of honeybee females can delay male development, which serves as a model for understanding the regulation of sex differentiation and male development in social insects by environmental factors.

Methods
For the raw data of table 1, honeybee queens were caged on an empty drone frame to lay eggs for 6 hrs. Eggs were grafted into queen and worker cells before hatching respectively. The eggs that remained in drone cells were used as controls. The weight of food remaining in three types of cells at 24 hrs, 48hrs, and 72 hrs was measured using an analytical balance

For the raw data of table 2-5, newly emerged drones were captured and weighed using an analytical balance (0.01 mg, AUY120, Shimazu Co. Ltd., Japan). Subsequently, drones were anesthetized by CO2, and their wing length and width, thorax width and head horizontal area were measured by a microscopic imaging system.

For the raw data of table 6-8, drones were dissected, and the testes, seminal vesicles and mucous glands were taken out and washed with saline solution and placed on glass slides. After removing redundant tissues, the location of the tissues was adjusted on glass slides, and the images were taken under the microscopic imaging system. The tunica testis of the testes was removed, and seminiferous tubules were divided into many small aggregations according to the method developed by Tavares et al (2003). The horizontal area of seminal vesical and mucous glands was measured by Image J, and the length of seminiferous tubules was measured by Troup view.