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

Ugajin A, Kiya T, Kunieda T, Ono M, Yoshida T, Kubo T - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

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.

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Related in: MedlinePlus

Identification and characterization of Acks, the Japanese honeybee kakusei homolog, as a non-coding IEG.(A) Overview of Acks cDNA and open reading frame (ORF) analysis. The yellow bar and black bar represent the Acks cDNA region highly conserved among the Acks and kakusei cDNAs and the region adjacent to the Acks cDNA, respectively. The arrow indicates the region corresponding to the putative neural activity-inducible Acks transcript. The orange bar indicates the region corresponding to sense and antisense probes used in the in situ hybridization. Horizontal boxes under the upper yellow and black bar indicate open reading frame analysis in each reading frame of the Acks cDNA, respectively. The blue and pink bars present in each box indicate positions of initiation and termination codons, respectively. Colored squares on the horizontal boxes indicate potential ORFs longer than 150b. The blue (in Frame 1) and red box (in Frame 1) indicate the longest ORF and the ORF conserved among Acks and kakusei cDNAs, respectively. (B) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under room temperature (25°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 30 min P<0.01; c, different from 45 min P<0.01; d, different from 60 min P<0.01; Tukey-Kramer's test). Sz-induced, seizure-induced. (C) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under the high temperature (46°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 15 min P<0.05; c, different from 90 min P<0.01; d, different from 150 min P<0.05; Tukey-Kramer's test).
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pone-0032902-g001: Identification and characterization of Acks, the Japanese honeybee kakusei homolog, as a non-coding IEG.(A) Overview of Acks cDNA and open reading frame (ORF) analysis. The yellow bar and black bar represent the Acks cDNA region highly conserved among the Acks and kakusei cDNAs and the region adjacent to the Acks cDNA, respectively. The arrow indicates the region corresponding to the putative neural activity-inducible Acks transcript. The orange bar indicates the region corresponding to sense and antisense probes used in the in situ hybridization. Horizontal boxes under the upper yellow and black bar indicate open reading frame analysis in each reading frame of the Acks cDNA, respectively. The blue and pink bars present in each box indicate positions of initiation and termination codons, respectively. Colored squares on the horizontal boxes indicate potential ORFs longer than 150b. The blue (in Frame 1) and red box (in Frame 1) indicate the longest ORF and the ORF conserved among Acks and kakusei cDNAs, respectively. (B) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under room temperature (25°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 30 min P<0.01; c, different from 45 min P<0.01; d, different from 60 min P<0.01; Tukey-Kramer's test). Sz-induced, seizure-induced. (C) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under the high temperature (46°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 15 min P<0.05; c, different from 90 min P<0.01; d, different from 150 min P<0.05; Tukey-Kramer's test).

Mentions: To map the active regions in the brains of the Japanese honeybee workers during the formation of a defensive bee ball, we intended to use the kakusei homolog as a neural activity marker. Because no kakusei homolog had been identified in any other animal species, including insects, however, we first amplified parts of kakusei cDNA from the Japanese honeybee using primers designed from the European honeybee kakusei cDNA sequences. Rapid amplification of the cDNA end (RACE) method was then used to identify the Apis cerana kakusei (Acks: Apis cerana kakusei) cDNA of approximately 7.8 kb in length. No 5′-upstream or 3′-downstream cDNA sequence was obtained by the RACE method, leading us to conclude that we obtained a full-length Acks cDNA. The nucleotide sequences of Acks cDNA of approximately 7.1 kb in length shared approximately 85% sequence identity with the European honeybee kakusei cDNA, and had additional unique sequences of approximately 700b at its 3′ end. The Acks cDNA contained six putative ORFs longer than 150b, and the longest ORF was 342b (Figure 1A). A comparison of the positions of the ORFs with those of kakusei cDNA revealed that one of the six ORFs is conserved among kakusei and Acks. This conserved ORF, however, was located at the 3′-region of the Acks cDNA, and its length was only 183b (Figure 1A). We previously showed that, in addition to the neural activity-inducible kakusei transcript, multiple neural activity-independent transcripts are constitutively expressed from the same kakusei locus, and the nucleotide sequences corresponding to +1925b to +5160b of the consensus kakusei cDNA are specifically contained in the neural activity-inducible kakusei-transcript [15]. Comparison of the structure and nucleotide sequences of Acks and kakusei cDNAs revealed that the nucleotide sequences corresponding to +1946b to +5175b of Acks cDNA was equivalent to the kakusei cDNA corresponding to the neural activity-inducible transcript (Figure 1A). These findings suggest that the cloned Acks cDNA also contained nucleotide sequences corresponding to the putative neural activity-inducible Acks transcript, and that, like the kakusei-transcript, the Acks transcript functions as a non-coding RNA.


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

Ugajin A, Kiya T, Kunieda T, Ono M, Yoshida T, Kubo T - PLoS ONE (2012)

Identification and characterization of Acks, the Japanese honeybee kakusei homolog, as a non-coding IEG.(A) Overview of Acks cDNA and open reading frame (ORF) analysis. The yellow bar and black bar represent the Acks cDNA region highly conserved among the Acks and kakusei cDNAs and the region adjacent to the Acks cDNA, respectively. The arrow indicates the region corresponding to the putative neural activity-inducible Acks transcript. The orange bar indicates the region corresponding to sense and antisense probes used in the in situ hybridization. Horizontal boxes under the upper yellow and black bar indicate open reading frame analysis in each reading frame of the Acks cDNA, respectively. The blue and pink bars present in each box indicate positions of initiation and termination codons, respectively. Colored squares on the horizontal boxes indicate potential ORFs longer than 150b. The blue (in Frame 1) and red box (in Frame 1) indicate the longest ORF and the ORF conserved among Acks and kakusei cDNAs, respectively. (B) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under room temperature (25°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 30 min P<0.01; c, different from 45 min P<0.01; d, different from 60 min P<0.01; Tukey-Kramer's test). Sz-induced, seizure-induced. (C) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under the high temperature (46°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 15 min P<0.05; c, different from 90 min P<0.01; d, different from 150 min P<0.05; Tukey-Kramer's test).
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Related In: Results  -  Collection

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pone-0032902-g001: Identification and characterization of Acks, the Japanese honeybee kakusei homolog, as a non-coding IEG.(A) Overview of Acks cDNA and open reading frame (ORF) analysis. The yellow bar and black bar represent the Acks cDNA region highly conserved among the Acks and kakusei cDNAs and the region adjacent to the Acks cDNA, respectively. The arrow indicates the region corresponding to the putative neural activity-inducible Acks transcript. The orange bar indicates the region corresponding to sense and antisense probes used in the in situ hybridization. Horizontal boxes under the upper yellow and black bar indicate open reading frame analysis in each reading frame of the Acks cDNA, respectively. The blue and pink bars present in each box indicate positions of initiation and termination codons, respectively. Colored squares on the horizontal boxes indicate potential ORFs longer than 150b. The blue (in Frame 1) and red box (in Frame 1) indicate the longest ORF and the ORF conserved among Acks and kakusei cDNAs, respectively. (B) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under room temperature (25°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 30 min P<0.01; c, different from 45 min P<0.01; d, different from 60 min P<0.01; Tukey-Kramer's test). Sz-induced, seizure-induced. (C) Time course of Acks expression level investigated by quantitative RT-PCR after seizure induction under the high temperature (46°C). Values are means ± SEM (a, different from 0 min P<0.01; b, different from 15 min P<0.05; c, different from 90 min P<0.01; d, different from 150 min P<0.05; Tukey-Kramer's test).
Mentions: To map the active regions in the brains of the Japanese honeybee workers during the formation of a defensive bee ball, we intended to use the kakusei homolog as a neural activity marker. Because no kakusei homolog had been identified in any other animal species, including insects, however, we first amplified parts of kakusei cDNA from the Japanese honeybee using primers designed from the European honeybee kakusei cDNA sequences. Rapid amplification of the cDNA end (RACE) method was then used to identify the Apis cerana kakusei (Acks: Apis cerana kakusei) cDNA of approximately 7.8 kb in length. No 5′-upstream or 3′-downstream cDNA sequence was obtained by the RACE method, leading us to conclude that we obtained a full-length Acks cDNA. The nucleotide sequences of Acks cDNA of approximately 7.1 kb in length shared approximately 85% sequence identity with the European honeybee kakusei cDNA, and had additional unique sequences of approximately 700b at its 3′ end. The Acks cDNA contained six putative ORFs longer than 150b, and the longest ORF was 342b (Figure 1A). A comparison of the positions of the ORFs with those of kakusei cDNA revealed that one of the six ORFs is conserved among kakusei and Acks. This conserved ORF, however, was located at the 3′-region of the Acks cDNA, and its length was only 183b (Figure 1A). We previously showed that, in addition to the neural activity-inducible kakusei transcript, multiple neural activity-independent transcripts are constitutively expressed from the same kakusei locus, and the nucleotide sequences corresponding to +1925b to +5160b of the consensus kakusei cDNA are specifically contained in the neural activity-inducible kakusei-transcript [15]. Comparison of the structure and nucleotide sequences of Acks and kakusei cDNAs revealed that the nucleotide sequences corresponding to +1946b to +5175b of Acks cDNA was equivalent to the kakusei cDNA corresponding to the neural activity-inducible transcript (Figure 1A). These findings suggest that the cloned Acks cDNA also contained nucleotide sequences corresponding to the putative neural activity-inducible Acks transcript, and that, like the kakusei-transcript, the Acks transcript functions as a non-coding RNA.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus