RNA-seq of antennal tissue of male and female honey bees (Apis mellifera) at 2 different time points of the day
In this RNA-seq study, we compared the antennal transcriptomes of sexually mature drones (males) and time-trained foragers (females) of Apis mellifera collected at different times of day and different activity states. The goals of our project was to provide a more comprehensive description of gene expression differences between drone and forager antennae. Most studies on insect antennal transcriptome still focus on identifying and reporting genes involved in odorant binding and detection. In contrast, we also aimed at identifying so far unrecognised molecules, not directly involved in odorant detection, but likely playing an important role in peripheral olfactory processing. We also wanted to explore whether daily changes in gene expression and correlations between gene expression levels and behavioral activity might be a fruitful approach to identify additional genes involved in odorant reception. Apis mellifera drones perform mating flight in the afternoon around 14:00 hour (h) in Bangalore, India. During their daily mating flight activity, drones were caught at the hive entrance and color marked on the thorax. On the next day color-marked drones were collected at two different time points: 9:00 (inactive) and 14:00 h (active).  Honey bee foragers can be trained to visit a food source at a specific time of the day. In this study, an A. mellifera colony was transferred in an enclosed outdoor flight cage to train the foragers to visit a feeder at a specific time of the day. A sucrose reward (1M sucrose solution) was presented either from 8:00 to 10:00 h (morning training) or from 13:00 to 15:00 h (afternoon training) for 10 consecutive days. On the 8th, 9th and 10th day of training, foragers visiting the feeder were marked on their thorax with different colors, one type of color each day, to identify the frequently visiting foragers. On the 11th day, the feeder was not presented and the foragers that had all 3 color marks were collected at 9:00 and 14:00 h.  All the samples were immediately flash frozen in liquid nitrogen. Collected samples were transferred from liquid nitrogen onto dry ice and the entire antennae (i.e. scape, pedicel and flagellum) were cut off. We pooled 10 antennae from 5 bees per sample and extracted total RNA using Trizol method. Antennal transcriptomes of drones (n=3 per time point), morning-trained foragers (n=2 per time point) and afternoon-trained foragers (n=2 per time point) were sequenced at 2 different time points (9:00 h and 14:00 h).