Abstract: 【Objective】 The objective of this study is to identify the hunger pheromone of honeybee queen and drone larvae and its biosynthetic pathway, which would enormously contribute to understanding of the mechanism of communication between adults and larvae in honey bees Apis mellifera. 【Method】Two- and four-day-old queen and drone larvae and their food were collected and divided into three groups: fed larvae, starving larvae and food. For the fed larvae they were lain on their relative foods prepared in advance, and for the starving larvae their foods were totally deprived. All samples were immediately put into 20 mL sealed glass bottles and were kept in an incubator under 35℃ for 45 min. Afterward, a needle trap system was employed to extract 10 mL gas from those bottles and the volatile chemicals were enriched in needles. The needles were injected into a gas chromatography-mass spectrometry system and the chemicals were dissociated by a high temperature of 250℃ for identifying the hunger pheromone of honeybee queen and drone larvae. RNA-Seq was used for identifying the biosynthetic pathway of their hunger pheromone and the expression of related genes. 【Result】Nine and ten chemicals were identified in drone larvae and queen larvae, respectively, in which queen larvae had one more chemical (2-heptanone) that was the highest royal jelly. E-β-ocimene was identified as the hunger-signal pheromone of queen and drone larvae, since their starving larvae had significantly more E-β-ocimene than related fed larvae. The E-β-ocimene released from queen and drone starving larvae was not significantly different at 2-day-old, but queen larvae released significantly less E-β-ocimene than drone larvae at 4-day-old. Other eight chemicals were myristic acid, palmitic acid, methyl palmitic ester, stearic acid, palmitoleic acid, pentadecanoic acid, acetic acid and ethyl acetate, but did not show a clear pattern that significantly more amount of these chemicals were detected in starving larvae released than fed larvae and food groups. Acetic acid and ethyl acetate were detected higher in food and fed larvae groups compared to starving larvae groups, indicating that these two chemicals may be from their food rather themselves. RNA-Seq analysis showed that there was a de novo E-β-ocimene biosynthetic pathway in queen and drone larvae via a mevalonate pathway. Nine genes such as Geranylgeranyl pyrophosphate synthase-like and Farnesyl pyrophosphate synthase were involved in this biosynthetic pathway, but all these genes were not significantly differentially expressed between starving larvae and their relative fed larvae. 【Conclusion】Honeybee queen and drone larvae both use E-β-ocimene as their hunger pheromone for food begging, and they have a de novo E-β-ocimene biosynthetic pathway. Further, nurse honeybees specifically use 2-heptanone to mark queen larvae for guiding other nurses. This study will enrich our understanding of the biological characteristics of honeybees.

Key words: Apis mellifera, hunger pheromone, E-β-ocimene, biosynthetic pathway, gene expression