Studies On Larval Hunger-Signal Pheromones And Larval Development In Honey Bee(Apis Mellifera)

Honeybees is a classical eusocial insect species and its' special biological characteristics have fascinated scientists for decades.This study used Apis mellifera ligustica(Aml) as experimental materials, and a needle trap and gas chromatography-mass spectrometry(GC-MS) system were employed for identifying the hunger pheromone of honeybee worker larvae. Nine chemicals were identified and one chemical, E-?-ocimene, was identified as the hunger-signal pheromone of worker larvae. Our results showed that starving larval treatments released significantly more E-?-ocimene than fed larval groups. Behavioural observation experiments showed that adding E-?-ocimene to empty cells increased the number of worker visits to those cells, and similarly adding E-?-ocimene to larvae increased worker visitation rate to the larvae, suggesting that E-?-ocimene is the candidate of larval hunger-signal pheromone. RNA-Seq analysis showed that there was a de novo E-?-ocimene biosynthetic pathway in worker larvae, and qRT-PCR identified 3 genes in the this pathway and they had the highest expression at a starving duration of 30 minutes.Two same systems, the needle trap and GC-MS, were used for identifying the hunger-signal pheromone of queen and drone larvae. Both queen and drone larvae used E-?-ocimene as their hunger-signal pheromone as well, as more E-?-ocimene were found in their starving larval groups compared to their fed groups. The E-?-ocimene from 2d starving queen, worker and drone larvae was not significantly different, but 4d starving queen larvae had significantly less E-?-ocimene than starving worker and drone larvae. Nine chemicals were found in drone and queen larvae same as worker larvae, however queen larvae had one more chemical(2-heptanone). RNA-Seq analysis also showed a same E-?-ocimene biosynthetic pathway in queen and drone larvae as worker larvae.RNA-Seq technology was utilized for comparing the gene expression of queen, drone and worker larvae at 2 and 4 day stages. The total number of expressed genes detected among 2- and 4-day old drone, queen and worker larvae was not significantly different, but the gene expression was significantly different among three castes. In detail, 2- and 4-day-old drone vs queen larval comparisons had more significantly differentially expressed genes(DEGs, 475 in 2d comparison and 687 in 4 d comparison respectively) than the drone and worker larval comparisons(197 and 604 in 2d and 4d comparsions respectively), and the worker and queen larval comparisons were the lowest(121 and 475). These results indicate that queen and drone larvae had the most DEGs and the developmental differentiation among three honeybee castes increases with larval age. Further, while comparing 2- and 4-day old larvae in a same larval type, drone larval comparison had dramatically less DEGs compared to the worker and queen larval comparisons(598 against 1190 and 1181), indicating that the haploid drones may have a different way on its developmental regulation of gene expression compared to the queen and workers. The co-expression analysis results showed that 2 d drone and worker larvae clustered firstly and then the 2 d queen larvae, whereas two female larave were more closer in gene expression at 4 day stage compared to 4d drone larvae. Many DEGs between drone larvae and two female larval types were involved in eye and muscle development, mTOR pathway, oocyte maturation, hormone biosynthesis, gene expression regulation and four signaling pathways, which are consistent with the caste differentiation among queen, workers and drones.