Honeybees organize a sophisticated society, and the workers transmit information about the location of food sources using a symbolic dance, known as ‘dance communication’. Recent studies indicate that workers integrate sensory information during foraging flight for dance communication. The neural mechanisms that account for this remarkable ability are, however, unknown. In the present study, we established a novel method to visualize neural activity in the honeybee brain using a novel immediate early gene, kakusei, as a marker of neural activity. The kakusei transcript was localized in the nuclei of brain neurons and did not encode an open reading frame, suggesting that it functions as a non-coding nuclear RNA. Using this method, we show that neural activity of a mushroom body neuron subtype, the small-type Kenyon cells, is prominently increased in the brains of dancer and forager honeybees. In contrast, the neural activity of the two mushroom body neuron subtypes, the small-and large-type Kenyon cells, is increased in the brains of re-orienting workers, which memorize their hive location during re-orienting flights. These findings demonstrate that the small-type Kenyon cell-preferential activity is associated with foraging behavior, suggesting its involvement in information integration during foraging flight, which is an essential basis for dance communication.

Many insects exhibit stereotypic instinctive behavior [1-3], but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates [4, 5]. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. Here, we identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, we mapped comprehensive activity patterns of the silkmoth brain in response to female sex pheromones. We found that Hr38 can also be used as a neural activity marker in the fly Drosophila melanogaster. Using Hr38, we constructed a neural activity map of the fly brain that partially overlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies.

The transcription factor FoxP2 is related to acoustic communication in vertebrates and, although widely expressed in various tissues, its mutations cause a speech disorder in humans and disrupt vocalization in mice. In honeybee colonies, workers transmit information about a food location using 'dance communication', which is a form of acoustic communication. We identified a honeybee FoxP2-homologue, AmFoxP, and investigated its expression in the honeybee brain to elucidate its possible role in dance communication. The relative abundance of AmFoxP mRNA in the worker brain increased during the first 4 days of adult life. In situ hybridization revealed AmFoxP expression around the optic lobes, central complex, dorsal lobes, and protocerebral lobes, which was not dependent on the caste or division of labour.

Anti-predator behaviors are essential to survival for most animals. The neural bases of such behaviors, however, remain largely unknown. Although honeybees commonly use their stingers to counterattack predators, the Japanese honeybee (Apis cerana japonica) uses a different strategy to fight against the giant hornet (Vespa mandarinia japonica). Instead of stinging the hornet, Japanese honeybees form a “hot defensive bee ball” by surrounding the hornet en masse, killing it with heat. The European honeybee (A. mellifera ligustica), on the other hand, does not exhibit this behavior, and their colonies are often destroyed by a hornet attack. In the present study, we attempted to analyze the neural basis of this behavior by mapping the active brain regions of Japanese honeybee workers during the formation of a hot defensive bee ball. First, we identified an A. cerana homolog (Acks = Apis cerana kakusei) of kakusei, an immediate early gene that we previously identified from A. mellifera, and showed that Acks has characteristics similar to kakusei and can be used to visualize active brain regions in A. cerana. Using Acks as a neural activity marker, we demonstrated that neural activity in the mushroom bodies, especially in Class II Kenyon cells, one subtype of mushroom body intrinsic neurons, and a restricted area between the dorsal lobes and the optic lobes was increased in the brains of Japanese honeybee workers involved in the formation of a hot defensive bee ball. In addition, workers exposed to 46°C heat also exhibited Acks expression patterns similar to those observed in the brains of workers involved in the formation of a hot defensive bee ball, suggesting that the neural activity observed in the brains of workers involved in the hot defensive bee ball mainly reflects thermal stimuli processing.