Detection of Neural Activity in the Brains of Japanese Honeybee Workers during the Formation of a “Hot Defensive Bee Ball”

Abstract

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.

Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

“…Quantitative RT-PCR was performed essentially as described previously [46]. After the OLs and the other brain regions (for Fig.…”

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

“…In general, honeybees use their stingers to counterattack an intruder [2], but Japanese honeybees exhibit a distinct defensive behavior, called “hot defensive bee ball formation”, when they are attacked by the giant hornet ( Vespa mandarinia japonica ) [48]. In Japan, giant hornets are the most formidable natural enemy of the honeybees and they continuously attack honeybee colonies to steal their larvae and pupae in autumn [18,48,49]. Because the exoskeleton of the giant hornets is so rigid that bee stings are ineffective, colonies of European honeybees are often destroyed.…”

“…This anti-predator behavior is a good example that selective pressure to avoid predation resulted in species-specific behavioral evolution [47]. To gain insights into how this behavior is acquired and evolved, as a first step, we initiated studies to elucidate the neural basis of this thermal defensive behavior by IEG mapping [18]. …”

“…No kakusei homologue was found in other species whose genomes were determined, suggesting that kakusei is a honeybee-specific noncoding RNA. We were, however, able to isolate a homologous gene from the Japanese honeybee, termed Acks ( Apis cerana kaku s ei ), with comprehensive reverse transcription-polymerase chain reaction experiments [18]. Like kakusei , Acks is rapidly expressed in a neural activity-dependent manner and the transcripts are localized exclusively in the nuclei.…”

“…Next, we describe that kakusei has various transcript variants, which includes an inducible isoform and constitutive isoforms, and discuss the possible neural function of kakusei [ 17]. Third, we discuss the isolation of a homologue of kakusei , termed Acks , from the Japanese honeybee ( Apis cerana ), and detection of neurons that are active in the workers fighting with the giant hornet, the natural enemy of Japanese honeybees [18]. …”