Taketoshi Kiya

Increased Neural Activity of a Mushroom Body Neuron Subtype in the Brains of Forager Honeybees

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.

Identification of novel Bombyxin genes from the genome of the silkmoth Bombyx mori and analysis of their expression

Insulin family peptide members play key roles in regulating growth, metabolism, and reproduction. Bombyxin is an insulin-related peptide of the silkmoth Bombyx mori. We analyzed the full genome of B. mori and identified five novel bombyxin families, V to Z. We characterized the genomic organization and chromosomal location of the novel bombyxin family genes. In contrast to previously identified bombyxin genes, bombyxin-V and -Z genes had intervening introns at almost the same positions as vertebrate insulin genes. We performed reverse transcription-polymerase chain reaction and in situ hybridization in different tissues and developmental stages to observe their temporal and spatial expression patterns. The newly identified bombyxin genes were expressed in diverse tissues: bombyxin-V, -W, and -Y mRNAs were expressed in the brain and bombyxin-X mRNA in fat bodies. Bombyxin-Y gene was expressed in both brain and ovary of larval stages. High level of bombyxin-Z gene expression in the follicular cells may suggest its function in reproduction. The presence of a short C-peptide domain and an extended A chain domain, and high expression of bombyxin-X gene in the fat body cells during non-feeding stages suggest its insulin-like growth factor-like function. These results suggest that the bombyxin genes originated from a common ancestral gene, similar to the vertebrate insulin gene, and evolved into a diverse gene family with multiple functions.

Inducible- and constitutive-type transcript variants of kakusei, a novel non-coding immediate early gene, in the honeybee brain

We previously identified a novel non‐coding immediate early gene, termed kakusei, from the honeybee (Apis mellifera) and used it as a marker to detect neural activity in the brains of foraging workers ( Kiya et al., 2007 ). Here, we investigated the detailed kakusei gene structure. Expression analysis revealed that, in addition to the neural activity‐inducible transcript variant, multiple neural activity‐independent transcript variants were constitutively expressed from the same kakusei locus. In situ hybridization revealed that constitutive‐type kakusei variants were detected in the whole brain and the RNA was localized predominantly in the neural nuclei, like the inducible‐type variant, suggesting the concerted action of inducible‐ and constitutive‐types of kakusei transcript variants on nuclear function.

Detection of neural activity in the brains of Japanese honeybee workers during the formation of a "hot defensive bee ball".

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.

Identification of kakusei, a Nuclear Non-Coding RNA, as an Immediate Early Gene from the Honeybee, and Its Application for Neuroethological Study

The honeybee is a social insect that exhibits various social behaviors. To elucidate the neural basis of honeybee behavior, we detected neural activity in freely-moving honeybee workers using an immediate early gene (IEG) that is expressed in a neural activity-dependent manner. In European honeybees (Apis mellifera), we identified a novel nuclear non-coding RNA, termed kakusei, as the first insect IEG, and revealed the neural activity pattern in foragers. In addition, we isolated a homologue of kakusei, termed Acks, from the Japanese honeybee (Apis cerana), and detected active neurons in workers fighting with the giant hornet.

Expression analysis of the FoxP homologue in the brain of the honeybee, Apis mellifera.

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.

Analysis of GABAergic and non-GABAergic neuron activity in the optic lobes of the forager and re-orienting worker honeybee (Apis mellifera L.).

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.

Dance Type and Flight Parameters Are Associated with Different Mushroom Body Neural Activities in Worker Honeybee Brains

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.

Glutamate receptors in the terminal nerve gonadotropin-releasing hormone neurons of the dwarf gourami (teleost)

The terminal nerve (TN)-gonadotropin-releasing hormone (GnRH) system has been suggested to function as a neuromodulatory system that regulates the motivational state of the animal. To investigate the synaptic control of activities of the TN-GnRH neurons, we analyzed electrophysiologically the type of glutamate receptors (GluRs) in the TN-GnRH neurons. By using various specific GluR agonists and antagonists, we found that they have ionotropic GluRs (iGluR; non-NMDAR and NMDAR) and group 3 metabotropic GluRs. However, in the combined presence of supramaximal concentration of iGluR blockers in the perfusing solution and the GDPbetaS in the patch pipette, there were still residual Glu-induced depolarizing responses. These results suggest the presence of a novel type of iGluRs, in addition to the conventional GluRs, in the TN-GnRH neurons.

Identification and characterization of a novel nuclear noncoding RNA, Fben-1, which is preferentially expressed in the higher brain center of the female silkworm moth, Bombyx mori.

Sexually dimorphic neural circuits are essential for the reproductive behavior. The molecular basis underling the sexual dimorphism, however, is still elusive in the brains of insects. To identify genes with sex-differential expression in the brain of silkworm moths, we performed fluorescent differential display screening and identified a novel gene, termed Fben-1 (Female-brain expressed noncoding RNA-1), whose expression is preferential to the female brain. Fben-1 cDNA sequences contained no significant open-reading frames and comprised a sex-differential transcript composition. Expression of Fben-1 was developmentally regulated and predominant in adult female moth brains. In situ hybridization revealed that Fben-1 is mainly expressed in the cells around the mushroom bodies, a higher brain center of the insect brain. In addition, Fben-1 transcripts were localized exclusively in the nuclei of these cells. This is the first report that a long nuclear noncoding RNA is expressed in a sex-differential manner in the higher center of insect brains. Our results suggest the possible involvement of nuclear noncoding RNA in sexually dimorphic brain functions.