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Background: Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking., Results: In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone., Conclusion: These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods.

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.

Background: Honeybee foragers can transmit the information concerning the location of food sources to their nestmates using dance communication. We previously used a novel immediate early gene, termed kakusei, to demonstrate that the neural activity of a specific mushroom body (MB) neuron subtype is preferentially enhanced in the forager brain. The sensory information related to this MB neuron activity, however, remained unclear., Methodology/principal Findings: Here, we used kakusei to analyze the relationship between MB neuron activity and types of foraging behavior. The number of kakusei-positive MB neurons was higher in the round dancers that had flown a short distance than in the waggle dancers that had flown a long distance. Furthermore, the amount of kakusei transcript in the MBs inversely related to the waggle-phase duration of the waggle dance, which correlates with the flight distance. Using a narrow tunnel whose inside was vertically or axially lined, we manipulated the pattern of visual input, which is received by the foragers during flight, and analysed kakusei expression. The amount of kakusei transcript in the MBs was related to the foraging frequency but not to the tunnel pattern. In contrast, the number of kakusei-positive MB neurons was affected by the tunnel patterns, but not related to foraging frequency., Conclusions/significance: These results suggest that the MB neuron activity depends on the foraging frequency, whereas the number of active MB neurons is related to the pattern of visual input received during foraging flight. Our results suggest that the foraging frequency and visual experience during foraging are associated with different MB neural activities.

Background: European honeybee (Apis mellifera L.) foragers have a highly developed visual system that is used for navigation. To clarify the neural basis underlying the highly sophisticated visual ability of foragers, we investigated the neural activity pattern of the optic lobes (OLs) in pollen-foragers and re-orienting bees, using the immediate early gene kakusei as a neural activity marker., Methodology/principal Findings: We performed double-in situ hybridization of kakusei and Amgad, the honeybee homolog of the GABA synthesizing enzyme GAD, to assess inhibitory neural activity. kakusei-related activity in GABAergic and non-GABAergic neurons was strongly upregulated in the OLs of the foragers and re-orienting bees, suggesting that both types of neurons are involved in visual information processing. GABAergic neuron activity was significantly higher than non-GABAergic neuron activity in a part of the OLs of only the forager, suggesting that unique information processing occurs in the OLs of foragers. In contrast, GABAergic neuron activity in the antennal lobe was significantly lower than that of GABAergic neurons in the OLs in the forager and re-orienting bees, suggesting that kakusei-related visual activity is dominant in the brains of these bees., Conclusions/significance: The present study provides the first evidence that GABAergic neurons are highly active in the OL neurons of free-moving honeybees and essential clue to reveal neural basis of the sophisticated visual ability that is equipped in the small and simple brain.

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.