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Ecdysteroid-dependent expression of the tweedle and peroxidase genes during adult cuticle formation in the honey bee, Apis mellifera.

Soares MP, Silva-Torres FA, Elias-Neto M, Nunes FM, Simões ZL, Bitondi MM - PLoS ONE (2011)

Bottom Line: Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome.The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland.Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

ABSTRACT
Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

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Relative quantification of AmelTwdl1, AmelTwd2 and Ampxd transcripts in different tissues of the Pbl pharate adult.The Amrp49 gene was used as endogenous control in real-time RT-PCR assays. The relative amount of transcripts is given by 2-ΔΔCT. Columns and bars represent means±SE of three independent samples prepared with each tissue. AmelTwdl1 transcript levels were significantly different among the tested tissues, with a higher expression in the gut (p = 0.002). Similarly, AmelTwdl2 showed a significantly higher expression in the gut than in integument and trachea (p = 0.010). In contrast, Ampxd showed a higher expression in integument than in the other tissues (p = 0.001). Statistical analysis was carried out with Jandel SigmaStat 3.1 software (Jandel Corporation, San Rafael, CA, USA). One Way Anova; post-hoc comparisons using Holm Sidak test p<0.05. Different letters above columns indicate statistical difference.
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pone-0020513-g005: Relative quantification of AmelTwdl1, AmelTwd2 and Ampxd transcripts in different tissues of the Pbl pharate adult.The Amrp49 gene was used as endogenous control in real-time RT-PCR assays. The relative amount of transcripts is given by 2-ΔΔCT. Columns and bars represent means±SE of three independent samples prepared with each tissue. AmelTwdl1 transcript levels were significantly different among the tested tissues, with a higher expression in the gut (p = 0.002). Similarly, AmelTwdl2 showed a significantly higher expression in the gut than in integument and trachea (p = 0.010). In contrast, Ampxd showed a higher expression in integument than in the other tissues (p = 0.001). Statistical analysis was carried out with Jandel SigmaStat 3.1 software (Jandel Corporation, San Rafael, CA, USA). One Way Anova; post-hoc comparisons using Holm Sidak test p<0.05. Different letters above columns indicate statistical difference.

Mentions: Real-time RT-PCR was used to quantify the levels of AmelTwdl1, AmelTwdl2 and Ampxd transcripts in the gut and trachea, and the expression in these tissues was compared to the expression in the thoracic integument. The levels of AmelTwd1 and AmelTwdl2 transcripts were significantly higher in the gut than in the thoracic integument (6.6- and 7.2-fold, respectively) and trachea (27.8- and 12.1-fold, respectively). In contrast, Ampxd showed the higher expression in the thoracic integument, with 7.8- and 2.2-fold higher transcript levels than in the gut and trachea, respectively (Figure 5).


Ecdysteroid-dependent expression of the tweedle and peroxidase genes during adult cuticle formation in the honey bee, Apis mellifera.

Soares MP, Silva-Torres FA, Elias-Neto M, Nunes FM, Simões ZL, Bitondi MM - PLoS ONE (2011)

Relative quantification of AmelTwdl1, AmelTwd2 and Ampxd transcripts in different tissues of the Pbl pharate adult.The Amrp49 gene was used as endogenous control in real-time RT-PCR assays. The relative amount of transcripts is given by 2-ΔΔCT. Columns and bars represent means±SE of three independent samples prepared with each tissue. AmelTwdl1 transcript levels were significantly different among the tested tissues, with a higher expression in the gut (p = 0.002). Similarly, AmelTwdl2 showed a significantly higher expression in the gut than in integument and trachea (p = 0.010). In contrast, Ampxd showed a higher expression in integument than in the other tissues (p = 0.001). Statistical analysis was carried out with Jandel SigmaStat 3.1 software (Jandel Corporation, San Rafael, CA, USA). One Way Anova; post-hoc comparisons using Holm Sidak test p<0.05. Different letters above columns indicate statistical difference.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105072&req=5

pone-0020513-g005: Relative quantification of AmelTwdl1, AmelTwd2 and Ampxd transcripts in different tissues of the Pbl pharate adult.The Amrp49 gene was used as endogenous control in real-time RT-PCR assays. The relative amount of transcripts is given by 2-ΔΔCT. Columns and bars represent means±SE of three independent samples prepared with each tissue. AmelTwdl1 transcript levels were significantly different among the tested tissues, with a higher expression in the gut (p = 0.002). Similarly, AmelTwdl2 showed a significantly higher expression in the gut than in integument and trachea (p = 0.010). In contrast, Ampxd showed a higher expression in integument than in the other tissues (p = 0.001). Statistical analysis was carried out with Jandel SigmaStat 3.1 software (Jandel Corporation, San Rafael, CA, USA). One Way Anova; post-hoc comparisons using Holm Sidak test p<0.05. Different letters above columns indicate statistical difference.
Mentions: Real-time RT-PCR was used to quantify the levels of AmelTwdl1, AmelTwdl2 and Ampxd transcripts in the gut and trachea, and the expression in these tissues was compared to the expression in the thoracic integument. The levels of AmelTwd1 and AmelTwdl2 transcripts were significantly higher in the gut than in the thoracic integument (6.6- and 7.2-fold, respectively) and trachea (27.8- and 12.1-fold, respectively). In contrast, Ampxd showed the higher expression in the thoracic integument, with 7.8- and 2.2-fold higher transcript levels than in the gut and trachea, respectively (Figure 5).

Bottom Line: Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome.The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland.Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

ABSTRACT
Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

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Related in: MedlinePlus