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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 nurse bees treated with 20-hydroxyecdysone.Nurse bees that were actively feeding the brood were collected from normal colonies. The 20E solution (1μl; 2.5μg/μl) was injected in the anterior aspect of the head. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 1 and 3 days after treatment. A total of two or three 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 Mblk-1 (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 20E-treated bees and control bees (*, p < 0.05; t-test).
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pone.0130206.g005: Quantification of gene expression in the HPGs of nurse bees treated with 20-hydroxyecdysone.Nurse bees that were actively feeding the brood were collected from normal colonies. The 20E solution (1μl; 2.5μg/μl) was injected in the anterior aspect of the head. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 1 and 3 days after treatment. A total of two or three 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 Mblk-1 (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 20E-treated bees and control bees (*, p < 0.05; t-test).

Mentions: On day 3 after 20E treatment, the mRNA level of Ambuffy, a nurse bee-selective gene, was significantly (~50%) lower in 20E-treated bees than in control bees (Welch’s t-test, p<0.01), while on day 1 after treatment, the Ambuffy mRNA level did not significantly differ between 20E-treated and control bees (Student’s t-test, p = 0.647) (Fig 5). The mRNA level of mrjp2, which is also a nurse bee-selective gene encoding a major royal jelly protein, was significantly (~70%) lower in 20E-treated bees than in control bees on day 1 after treatment (Welch’s t-test, p<0.01), and approximately 60% lower in 20E-treated bees than in control bees on day 3 after the treatment, although the latter decrease was not statistically significant (Welch’s t-test, p = 0.0786) (Fig 5). The changes in these gene expression levels appeared to mimic the changes in the expression levels of Ambuffy and mrjp2, both of which are nurse bee-selective genes whose expression decreases in association with the role change of the workers. There are some possible explanations for the differential effects of 20E on the expression of Ambuffy and mrjp2. For example, it might be that 20E temporarily represses mrjp2 expression and the resulting decline in mrjp2 expression leads to a decrease in Ambuffy expression while mrjp2 expression resumes. Another possibility is that repression of Ambuffy expression requires developmentally regulated factor(s) other than 20E, such as JH whose titer in the hemolymph increases over the worker’s lifetime, because gene expression was analyzed 3 days after 20E treatment and as the bees got older.


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 nurse bees treated with 20-hydroxyecdysone.Nurse bees that were actively feeding the brood were collected from normal colonies. The 20E solution (1μl; 2.5μg/μl) was injected in the anterior aspect of the head. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 1 and 3 days after treatment. A total of two or three 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 Mblk-1 (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 20E-treated bees and control bees (*, p < 0.05; t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470657&req=5

pone.0130206.g005: Quantification of gene expression in the HPGs of nurse bees treated with 20-hydroxyecdysone.Nurse bees that were actively feeding the brood were collected from normal colonies. The 20E solution (1μl; 2.5μg/μl) was injected in the anterior aspect of the head. HPGs were dissected out from worker heads and subjected to quantitative RT-PCR analysis at 1 and 3 days after treatment. A total of two or three 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 Mblk-1 (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 20E-treated bees and control bees (*, p < 0.05; t-test).
Mentions: On day 3 after 20E treatment, the mRNA level of Ambuffy, a nurse bee-selective gene, was significantly (~50%) lower in 20E-treated bees than in control bees (Welch’s t-test, p<0.01), while on day 1 after treatment, the Ambuffy mRNA level did not significantly differ between 20E-treated and control bees (Student’s t-test, p = 0.647) (Fig 5). The mRNA level of mrjp2, which is also a nurse bee-selective gene encoding a major royal jelly protein, was significantly (~70%) lower in 20E-treated bees than in control bees on day 1 after treatment (Welch’s t-test, p<0.01), and approximately 60% lower in 20E-treated bees than in control bees on day 3 after the treatment, although the latter decrease was not statistically significant (Welch’s t-test, p = 0.0786) (Fig 5). The changes in these gene expression levels appeared to mimic the changes in the expression levels of Ambuffy and mrjp2, both of which are nurse bee-selective genes whose expression decreases in association with the role change of the workers. There are some possible explanations for the differential effects of 20E on the expression of Ambuffy and mrjp2. For example, it might be that 20E temporarily represses mrjp2 expression and the resulting decline in mrjp2 expression leads to a decrease in Ambuffy expression while mrjp2 expression resumes. Another possibility is that repression of Ambuffy expression requires developmentally regulated factor(s) other than 20E, such as JH whose titer in the hemolymph increases over the worker’s lifetime, because gene expression was analyzed 3 days after 20E treatment and as the bees got older.

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.