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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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Tweedle: gene structures and alignment of insect tweedle proteins.(A) Schematic representation of the AmelTwdl1 and AmelTwdl2 genes. Initiation and termination codons are indicated at the left and right of the figure, respectively. Exons and introns are indicated by boxes and lines, respectively, and the number of nucleotides is shown. The direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of AmelTwdl1 (ACJ38118.1) and AmelTwdl2 (ADK73965.2) amino acid sequences with other Tweedle protein sequences from B. mori, BmGRP2 (BAE06189.1), and D. melanogaster, TwdlT (AAF56656.1) and TwdlE (AAF52571.2). The four conserved blocks of amino acids are marked in dark gray. The signal peptide region is underlined in all sequences. In light gray is a region of BmGRP2 sequence that was used by Zhong et al. (see ref. [40]) to synthesize a peptide for antibody production. This antibody recognized the AmelTwdl1 protein (see Fig. 3 and corresponding text in Results section). Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.
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pone-0020513-g001: Tweedle: gene structures and alignment of insect tweedle proteins.(A) Schematic representation of the AmelTwdl1 and AmelTwdl2 genes. Initiation and termination codons are indicated at the left and right of the figure, respectively. Exons and introns are indicated by boxes and lines, respectively, and the number of nucleotides is shown. The direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of AmelTwdl1 (ACJ38118.1) and AmelTwdl2 (ADK73965.2) amino acid sequences with other Tweedle protein sequences from B. mori, BmGRP2 (BAE06189.1), and D. melanogaster, TwdlT (AAF56656.1) and TwdlE (AAF52571.2). The four conserved blocks of amino acids are marked in dark gray. The signal peptide region is underlined in all sequences. In light gray is a region of BmGRP2 sequence that was used by Zhong et al. (see ref. [40]) to synthesize a peptide for antibody production. This antibody recognized the AmelTwdl1 protein (see Fig. 3 and corresponding text in Results section). Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.

Mentions: Using the B. mori BmGRP2 deduced protein (Accession number BAE06189.1, [40]) as a query in BLAST searches, we identified the GB19234 sequence in the genomic region GroupUn.1241. Specific primers were designed for this honey bee sequence, and total RNA was extracted from the thoracic integument. In a reverse transcription procedure, the corresponding cDNA was synthesized, amplified and sequenced. The sequenced cDNA was compared against the corresponding annotated gene in silico using the Artemis platform, revealing the exon/intron boundaries (Figure 1A). The CDS spans 999 nucleotides (stop codon included) distributed among 3 exons. Only the last four nucleotides at the 3′ region and the stop codon were not validated by cDNA sequencing. The conceptual primary protein sequence consists of 332 amino acids. It has a molecular mass of 30.81 kDa and a pI value of 9.2. The N-terminal signal peptide spans 47 amino acids (see in File S2 the nucleotides validated by sequencing, the deduced amino acid sequence, and the signal peptide region). The deduced protein contains the four conserved blocks of amino acids (Blocks I, II, III and IV) that typically characterize the Tweedle CP family, as observed in the ClustalW alignment with Tweedle proteins from other species of insects shown in Figure 1B. This honey bee gene was then named AmelTwdl1 (Amel was used to conserve the nomenclature given to the A. mellifera CPs in the CuticleDB; Twdl is the abbreviation given to tweedle genes first described in Drosophila), and the GB19234 sequence prediction was validated as a cuticle protein gene. The AmelTwdl1 protein shares 58% similarity (ClustaW score) with BmGRP2 and 60% and 48% similarities with the TwdlT (AAF56656.1) and TwdlE (AAF52571.2) proteins from D. melanogaster, respectively [49].


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)

Tweedle: gene structures and alignment of insect tweedle proteins.(A) Schematic representation of the AmelTwdl1 and AmelTwdl2 genes. Initiation and termination codons are indicated at the left and right of the figure, respectively. Exons and introns are indicated by boxes and lines, respectively, and the number of nucleotides is shown. The direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of AmelTwdl1 (ACJ38118.1) and AmelTwdl2 (ADK73965.2) amino acid sequences with other Tweedle protein sequences from B. mori, BmGRP2 (BAE06189.1), and D. melanogaster, TwdlT (AAF56656.1) and TwdlE (AAF52571.2). The four conserved blocks of amino acids are marked in dark gray. The signal peptide region is underlined in all sequences. In light gray is a region of BmGRP2 sequence that was used by Zhong et al. (see ref. [40]) to synthesize a peptide for antibody production. This antibody recognized the AmelTwdl1 protein (see Fig. 3 and corresponding text in Results section). Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105072&req=5

pone-0020513-g001: Tweedle: gene structures and alignment of insect tweedle proteins.(A) Schematic representation of the AmelTwdl1 and AmelTwdl2 genes. Initiation and termination codons are indicated at the left and right of the figure, respectively. Exons and introns are indicated by boxes and lines, respectively, and the number of nucleotides is shown. The direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of AmelTwdl1 (ACJ38118.1) and AmelTwdl2 (ADK73965.2) amino acid sequences with other Tweedle protein sequences from B. mori, BmGRP2 (BAE06189.1), and D. melanogaster, TwdlT (AAF56656.1) and TwdlE (AAF52571.2). The four conserved blocks of amino acids are marked in dark gray. The signal peptide region is underlined in all sequences. In light gray is a region of BmGRP2 sequence that was used by Zhong et al. (see ref. [40]) to synthesize a peptide for antibody production. This antibody recognized the AmelTwdl1 protein (see Fig. 3 and corresponding text in Results section). Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.
Mentions: Using the B. mori BmGRP2 deduced protein (Accession number BAE06189.1, [40]) as a query in BLAST searches, we identified the GB19234 sequence in the genomic region GroupUn.1241. Specific primers were designed for this honey bee sequence, and total RNA was extracted from the thoracic integument. In a reverse transcription procedure, the corresponding cDNA was synthesized, amplified and sequenced. The sequenced cDNA was compared against the corresponding annotated gene in silico using the Artemis platform, revealing the exon/intron boundaries (Figure 1A). The CDS spans 999 nucleotides (stop codon included) distributed among 3 exons. Only the last four nucleotides at the 3′ region and the stop codon were not validated by cDNA sequencing. The conceptual primary protein sequence consists of 332 amino acids. It has a molecular mass of 30.81 kDa and a pI value of 9.2. The N-terminal signal peptide spans 47 amino acids (see in File S2 the nucleotides validated by sequencing, the deduced amino acid sequence, and the signal peptide region). The deduced protein contains the four conserved blocks of amino acids (Blocks I, II, III and IV) that typically characterize the Tweedle CP family, as observed in the ClustalW alignment with Tweedle proteins from other species of insects shown in Figure 1B. This honey bee gene was then named AmelTwdl1 (Amel was used to conserve the nomenclature given to the A. mellifera CPs in the CuticleDB; Twdl is the abbreviation given to tweedle genes first described in Drosophila), and the GB19234 sequence prediction was validated as a cuticle protein gene. The AmelTwdl1 protein shares 58% similarity (ClustaW score) with BmGRP2 and 60% and 48% similarities with the TwdlT (AAF56656.1) and TwdlE (AAF52571.2) proteins from D. melanogaster, respectively [49].

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.

Show MeSH
Related in: MedlinePlus