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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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Peroxidase: gene structure and alignment of insect peroxidase proteins.(A) Schematic representation of the peroxidase gene from A. mellifera. Initiation and termination codons are indicated, as well as exons (boxes) and introns (lines). The number of nucleotides is shown, and the direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of peroxidase sequences from A. mellifera (AmPXD, ADE45321.2), Culex quinquefasciatus (CqPXD, EDS26535.1) and Aedes aegypti (AaPXD, EAT46477.1). The signal peptide region was underlined in the A. mellifera and C. quinquefasciatus sequences. The region containing the Animal haem peroxidase domain (pfam03098) is marked in grey. Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.
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pone-0020513-g002: Peroxidase: gene structure and alignment of insect peroxidase proteins.(A) Schematic representation of the peroxidase gene from A. mellifera. Initiation and termination codons are indicated, as well as exons (boxes) and introns (lines). The number of nucleotides is shown, and the direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of peroxidase sequences from A. mellifera (AmPXD, ADE45321.2), Culex quinquefasciatus (CqPXD, EDS26535.1) and Aedes aegypti (AaPXD, EAT46477.1). The signal peptide region was underlined in the A. mellifera and C. quinquefasciatus sequences. The region containing the Animal haem peroxidase domain (pfam03098) is marked in grey. Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.

Mentions: BLASTP analysis using the predicted peroxidases of Aedes aegypti (EAT46477.1) and Culex quinquefasciatus (EDS26535.1) failed to identify a peroxidase gene prediction in the Official Gene Set pre_release2 database. However, we verified that in the honey bee genome version 4.0, both dipteran genes aligned to two regions, Group4.3 and GroupUn.1247. To annotate and determine the structure of the honey bee peroxidase gene, both linkage groups were reoriented and merged. These two orthologous sequences enabled us to manually predict a gene model. Gene sequencing using primers (primers Ampxd f2 and r2, see File S1) flanking regions mapped on both linkage groups, Group4.3 and GroupUn.1247, confirmed that these groups are indeed continuous. The identified peroxidase gene spans ∼21 kb of genomic DNA. The annotated CDS has 1977 nucleotides (stop codon included) and is separated into 13 exons (Figure 2A) that potentially encode a protein of 658 amino acids. Expected canonical splice sites (conforming to the GT/AG rule) were found at all exon/intron boundaries. The CDS was partially sequenced, with 1403 of the 1977 nucleotides identified, validating 7 entire and 2 partial annotated exons. These sequence data were submitted to GenBank under accession number GU785071.2 (ADE45321.2 for its conceptual translation product). The Animal haem peroxidase domain (pfam03098) spanning a region of 541 amino acids was identified in the deduced translation product. The predicted N-terminal signal peptide includes 26 residues (see in File S4 the CDS region confirmed by sequencing, the translated protein sequence, and the signal peptide). The molecular mass of the annotated protein is 75.08 kDa, and its pI value is 5.8. Leucine is the most abundant amino acid in the deduced peroxidase protein, comprising 10.3% of the amino acid residues. Other amino acids are present at a lower abundance, which ranges from 1.5 to 7.0%. The honey bee peroxidase protein shows 80% and 79% similarity with the peroxidases of A. aegypti and C. quinquefasciatus, respectively (ClustalW score) (Figure 2B).


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)

Peroxidase: gene structure and alignment of insect peroxidase proteins.(A) Schematic representation of the peroxidase gene from A. mellifera. Initiation and termination codons are indicated, as well as exons (boxes) and introns (lines). The number of nucleotides is shown, and the direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of peroxidase sequences from A. mellifera (AmPXD, ADE45321.2), Culex quinquefasciatus (CqPXD, EDS26535.1) and Aedes aegypti (AaPXD, EAT46477.1). The signal peptide region was underlined in the A. mellifera and C. quinquefasciatus sequences. The region containing the Animal haem peroxidase domain (pfam03098) is marked in grey. Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105072&req=5

pone-0020513-g002: Peroxidase: gene structure and alignment of insect peroxidase proteins.(A) Schematic representation of the peroxidase gene from A. mellifera. Initiation and termination codons are indicated, as well as exons (boxes) and introns (lines). The number of nucleotides is shown, and the direction of transcription is indicated by an arrow. (B) Alignment (ClustalW 2) of peroxidase sequences from A. mellifera (AmPXD, ADE45321.2), Culex quinquefasciatus (CqPXD, EDS26535.1) and Aedes aegypti (AaPXD, EAT46477.1). The signal peptide region was underlined in the A. mellifera and C. quinquefasciatus sequences. The region containing the Animal haem peroxidase domain (pfam03098) is marked in grey. Asterisks, colons and dots represent identical amino acid residues, strong- and weak-conservative substitutions, respectively.
Mentions: BLASTP analysis using the predicted peroxidases of Aedes aegypti (EAT46477.1) and Culex quinquefasciatus (EDS26535.1) failed to identify a peroxidase gene prediction in the Official Gene Set pre_release2 database. However, we verified that in the honey bee genome version 4.0, both dipteran genes aligned to two regions, Group4.3 and GroupUn.1247. To annotate and determine the structure of the honey bee peroxidase gene, both linkage groups were reoriented and merged. These two orthologous sequences enabled us to manually predict a gene model. Gene sequencing using primers (primers Ampxd f2 and r2, see File S1) flanking regions mapped on both linkage groups, Group4.3 and GroupUn.1247, confirmed that these groups are indeed continuous. The identified peroxidase gene spans ∼21 kb of genomic DNA. The annotated CDS has 1977 nucleotides (stop codon included) and is separated into 13 exons (Figure 2A) that potentially encode a protein of 658 amino acids. Expected canonical splice sites (conforming to the GT/AG rule) were found at all exon/intron boundaries. The CDS was partially sequenced, with 1403 of the 1977 nucleotides identified, validating 7 entire and 2 partial annotated exons. These sequence data were submitted to GenBank under accession number GU785071.2 (ADE45321.2 for its conceptual translation product). The Animal haem peroxidase domain (pfam03098) spanning a region of 541 amino acids was identified in the deduced translation product. The predicted N-terminal signal peptide includes 26 residues (see in File S4 the CDS region confirmed by sequencing, the translated protein sequence, and the signal peptide). The molecular mass of the annotated protein is 75.08 kDa, and its pI value is 5.8. Leucine is the most abundant amino acid in the deduced peroxidase protein, comprising 10.3% of the amino acid residues. Other amino acids are present at a lower abundance, which ranges from 1.5 to 7.0%. The honey bee peroxidase protein shows 80% and 79% similarity with the peroxidases of A. aegypti and C. quinquefasciatus, respectively (ClustalW score) (Figure 2B).

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