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Changes in the Gene Expression Profiles of the Hypopharyngeal Gland of Worker Honeybees in Association with Worker Behavior and Hormonal Factors.

Ueno T, Takeuchi H, Kawasaki K, Kubo T - PLoS ONE (2015)

Bottom Line: In the present study, to clarify the molecular mechanisms that coordinate HPG physiology with worker behavior, we first analyzed whether Ambuffy, AmMMP1, mrjp2 (a gene encoding one of major royal jelly protein isoforms), and Hbg3 (a gene encoding α-glucosidase III) expression, is associated with worker behavior in 'single-cohort colonies' where workers of almost the same age perform different tasks.Expression of these genes correlated with the worker's role, while controlling for age, indicating their regulation associated with the worker's behavior.Our findings suggest that both ecdysone- and JH-signaling cooperatively regulate the physiological state of the HPGs in association with the worker's behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan; Faculty of Pharmaceutical Sciences, Doshisha Women's College, Kyotanabe, Kyoto, 610-0395, Japan.

ABSTRACT
The hypopharyngeal glands (HPGs) of worker honeybees undergo physiological changes along with the age-dependent role change from nursing to foraging: nurse bee HPGs secrete mainly major royal jelly proteins, whereas forager HPGs secrete mainly α-glucosidase III, which converts the sucrose in the nectar into glucose and fructose. We previously identified two other genes, Apis mellifera buffy (Ambuffy) and Apis mellifera matrix metalloproteinase 1 (AmMMP1), with enriched expression in nurse bee and forager HPGs, respectively. In the present study, to clarify the molecular mechanisms that coordinate HPG physiology with worker behavior, we first analyzed whether Ambuffy, AmMMP1, mrjp2 (a gene encoding one of major royal jelly protein isoforms), and Hbg3 (a gene encoding α-glucosidase III) expression, is associated with worker behavior in 'single-cohort colonies' where workers of almost the same age perform different tasks. Expression of these genes correlated with the worker's role, while controlling for age, indicating their regulation associated with the worker's behavior. Associated gene expression suggested the possible involvement of some hormonal factors in its regulation. We therefore examined the relationship between ecdysone- and juvenile hormone (JH)-signaling, and the expression profiles of these 'indicator' genes (nurse bee HPG-selective genes: mrjp2 and Ambuffy, and forager HPG-selective genes: Hbg3 and AmMMP1). Expression of both ecdysone-regulated genes (ecdysone receptor, mushroom body large type Kenyon cell specific protein-1, and E74) and JH-regulated genes (Methoprene tolerant and Krüppel homolog 1) was higher in the forager HPGs than in the nurse bee HPGs, suggesting the possible roles of ecdysone- and JH-regulated genes in worker HPGs. Furthermore, 20-hydroxyecdysone-treatment repressed both nurse bee- and forager-selective gene expression, whereas methoprene-treatment enhanced the expression of forager-selective genes and repressed nurse bee-selective genes in the HPGs. Our findings suggest that both ecdysone- and JH-signaling cooperatively regulate the physiological state of the HPGs in association with the worker's behavior.

No MeSH data available.


Quantification of gene expression in the HPGs of worker honeybees treated with methoprene.Methoprene (250 μg) dissolved in acetone was applied to the heads of 6-day old workers. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 7 days after treatment. A total of two trials were performed to confirm the reproducibility. Gene transcripts were quantified from pooled samples obtained from all trials. Relative mRNA levels of Ambuffy (A), mrjp2 (B), AmMMP1 (C), Hbg3 (D), EcR (E), and Met (F) are indicated with the standard error, with the amount of mRNA in the HPGs of control bees defined as 1. Transcript amounts were normalized with that of elongation factor 1α-F2 (EF1α-F2). Asterisks indicate significant differences between methoprene-treated bees and control bees (*, p < 0.05; t-test).
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pone.0130206.g007: Quantification of gene expression in the HPGs of worker honeybees treated with methoprene.Methoprene (250 μg) dissolved in acetone was applied to the heads of 6-day old workers. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 7 days after treatment. A total of two trials were performed to confirm the reproducibility. Gene transcripts were quantified from pooled samples obtained from all trials. Relative mRNA levels of Ambuffy (A), mrjp2 (B), AmMMP1 (C), Hbg3 (D), EcR (E), and Met (F) are indicated with the standard error, with the amount of mRNA in the HPGs of control bees defined as 1. Transcript amounts were normalized with that of elongation factor 1α-F2 (EF1α-F2). Asterisks indicate significant differences between methoprene-treated bees and control bees (*, p < 0.05; t-test).

Mentions: Quantitative RT-PCR analysis revealed that the mRNA level of Ambuffy, a nurse bee-selective gene, was approximately 30% lower in methoprene-treated bees than in control bees (Student’s t-test, p = 0.173), while the mRNA level of mrjp2, which was also a nurse bee-selective gene, was significantly (~65%) lower in methoprene-treated bees than in control bees (Welch’s t-test, p<0.05) (Fig 7). On the other hand, the mRNA levels of AmMMP1 and Hbg3, which were forager-selective genes, were significantly higher (2.5 to 5.5-fold) in the methoprene-treated bees than in the control bees (Welch’s t-test, p<0.001 and p<0.001, respectively) (Fig 7). The changes in these gene expression levels were similar to the changes of the gene expression levels in HPGs in association with the role change from nursing to foraging in normal colonies. In addition, we examined the gene transcripts of EcR and Met, which are ecdysone- and JH-related genes, respectively. The results indicated that the mRNA levels of these two genes were approximately 2.0-fold higher in methoprene-treated bees than in control bees, as well as the mRNA levels of AmMMP1 and Hbg3, although the decreases were not statistically significant (Welch’s t-test, p = 0.111 and Student’s t-test, p = 0.103) (Fig 7). These findings suggest that JH downregulates expression levels of nurse bee-selective genes, whereas upregulates the expression levels of forager-selective genes in the HPGs. Finally, it should be emphasized that we did not examine the effect of hormone-treatment on worker behavior in the present study because hormone-injected workers were expelled from their colonies by the other workers. Therefore, our experiments only confirmed gene expression changes and did not address causation of the worker behavior.


Changes in the Gene Expression Profiles of the Hypopharyngeal Gland of Worker Honeybees in Association with Worker Behavior and Hormonal Factors.

Ueno T, Takeuchi H, Kawasaki K, Kubo T - PLoS ONE (2015)

Quantification of gene expression in the HPGs of worker honeybees treated with methoprene.Methoprene (250 μg) dissolved in acetone was applied to the heads of 6-day old workers. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 7 days after treatment. A total of two trials were performed to confirm the reproducibility. Gene transcripts were quantified from pooled samples obtained from all trials. Relative mRNA levels of Ambuffy (A), mrjp2 (B), AmMMP1 (C), Hbg3 (D), EcR (E), and Met (F) are indicated with the standard error, with the amount of mRNA in the HPGs of control bees defined as 1. Transcript amounts were normalized with that of elongation factor 1α-F2 (EF1α-F2). Asterisks indicate significant differences between methoprene-treated bees and control bees (*, p < 0.05; t-test).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470657&req=5

pone.0130206.g007: Quantification of gene expression in the HPGs of worker honeybees treated with methoprene.Methoprene (250 μg) dissolved in acetone was applied to the heads of 6-day old workers. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 7 days after treatment. A total of two trials were performed to confirm the reproducibility. Gene transcripts were quantified from pooled samples obtained from all trials. Relative mRNA levels of Ambuffy (A), mrjp2 (B), AmMMP1 (C), Hbg3 (D), EcR (E), and Met (F) are indicated with the standard error, with the amount of mRNA in the HPGs of control bees defined as 1. Transcript amounts were normalized with that of elongation factor 1α-F2 (EF1α-F2). Asterisks indicate significant differences between methoprene-treated bees and control bees (*, p < 0.05; t-test).
Mentions: Quantitative RT-PCR analysis revealed that the mRNA level of Ambuffy, a nurse bee-selective gene, was approximately 30% lower in methoprene-treated bees than in control bees (Student’s t-test, p = 0.173), while the mRNA level of mrjp2, which was also a nurse bee-selective gene, was significantly (~65%) lower in methoprene-treated bees than in control bees (Welch’s t-test, p<0.05) (Fig 7). On the other hand, the mRNA levels of AmMMP1 and Hbg3, which were forager-selective genes, were significantly higher (2.5 to 5.5-fold) in the methoprene-treated bees than in the control bees (Welch’s t-test, p<0.001 and p<0.001, respectively) (Fig 7). The changes in these gene expression levels were similar to the changes of the gene expression levels in HPGs in association with the role change from nursing to foraging in normal colonies. In addition, we examined the gene transcripts of EcR and Met, which are ecdysone- and JH-related genes, respectively. The results indicated that the mRNA levels of these two genes were approximately 2.0-fold higher in methoprene-treated bees than in control bees, as well as the mRNA levels of AmMMP1 and Hbg3, although the decreases were not statistically significant (Welch’s t-test, p = 0.111 and Student’s t-test, p = 0.103) (Fig 7). These findings suggest that JH downregulates expression levels of nurse bee-selective genes, whereas upregulates the expression levels of forager-selective genes in the HPGs. Finally, it should be emphasized that we did not examine the effect of hormone-treatment on worker behavior in the present study because hormone-injected workers were expelled from their colonies by the other workers. Therefore, our experiments only confirmed gene expression changes and did not address causation of the worker behavior.

Bottom Line: In the present study, to clarify the molecular mechanisms that coordinate HPG physiology with worker behavior, we first analyzed whether Ambuffy, AmMMP1, mrjp2 (a gene encoding one of major royal jelly protein isoforms), and Hbg3 (a gene encoding α-glucosidase III) expression, is associated with worker behavior in 'single-cohort colonies' where workers of almost the same age perform different tasks.Expression of these genes correlated with the worker's role, while controlling for age, indicating their regulation associated with the worker's behavior.Our findings suggest that both ecdysone- and JH-signaling cooperatively regulate the physiological state of the HPGs in association with the worker's behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan; Faculty of Pharmaceutical Sciences, Doshisha Women's College, Kyotanabe, Kyoto, 610-0395, Japan.

ABSTRACT
The hypopharyngeal glands (HPGs) of worker honeybees undergo physiological changes along with the age-dependent role change from nursing to foraging: nurse bee HPGs secrete mainly major royal jelly proteins, whereas forager HPGs secrete mainly α-glucosidase III, which converts the sucrose in the nectar into glucose and fructose. We previously identified two other genes, Apis mellifera buffy (Ambuffy) and Apis mellifera matrix metalloproteinase 1 (AmMMP1), with enriched expression in nurse bee and forager HPGs, respectively. In the present study, to clarify the molecular mechanisms that coordinate HPG physiology with worker behavior, we first analyzed whether Ambuffy, AmMMP1, mrjp2 (a gene encoding one of major royal jelly protein isoforms), and Hbg3 (a gene encoding α-glucosidase III) expression, is associated with worker behavior in 'single-cohort colonies' where workers of almost the same age perform different tasks. Expression of these genes correlated with the worker's role, while controlling for age, indicating their regulation associated with the worker's behavior. Associated gene expression suggested the possible involvement of some hormonal factors in its regulation. We therefore examined the relationship between ecdysone- and juvenile hormone (JH)-signaling, and the expression profiles of these 'indicator' genes (nurse bee HPG-selective genes: mrjp2 and Ambuffy, and forager HPG-selective genes: Hbg3 and AmMMP1). Expression of both ecdysone-regulated genes (ecdysone receptor, mushroom body large type Kenyon cell specific protein-1, and E74) and JH-regulated genes (Methoprene tolerant and Krüppel homolog 1) was higher in the forager HPGs than in the nurse bee HPGs, suggesting the possible roles of ecdysone- and JH-regulated genes in worker HPGs. Furthermore, 20-hydroxyecdysone-treatment repressed both nurse bee- and forager-selective gene expression, whereas methoprene-treatment enhanced the expression of forager-selective genes and repressed nurse bee-selective genes in the HPGs. Our findings suggest that both ecdysone- and JH-signaling cooperatively regulate the physiological state of the HPGs in association with the worker's behavior.

No MeSH data available.