Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

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.

Genes involved in thoracic exoskeleton formation during the pupal-to-adult molt in a social insect model, Apis mellifera

“…However, except for extensive studies on cuticular gene expression in Anopheles gambiae [9-11], few putative CP genes in annotated insect genomes have been experimentally validated (by determining transcript tissue-specificity or developmental profiles). In A. mellifera , only six of the 47 CP genes screened in the annotated genome [8] have been validated, including the AmelCPR14 gene [12] (bearing the chitin-binding R&R Consensus [13]), three genes in the Apidermins class [14], and the Tweedle class genes, AmelTwdl1 and AmelTwdl2 [15]. …”

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera

“…In parallel, genes acting in the integument, whose expression patterns were previously characterized in our laboratory, were herein investigated as potential targets of bursicon. These genes encode two cuticular proteins from the CPR family, AmCPR14 [14] and AmCPR3 [9], two Tweedle proteins, AmTwdl1 and AmTwdl2 [8], two apidermins, Amapd2 and Amapd3 [9, 11], and three enzymes potentially acting in the cuticular melanization/sclerotization pathway, namely, tyrosine hydroxylase ( Amth ) and dopa decarboxylase ( Amddc ) and a peroxidase ( Ampxd ) [8]. The accession numbers for all these genes are given in S1 Table.…”

“…The A . mellifera genome contains two genes encoding Tweedle proteins [8, 9]. Members of this protein class share four conserved blocks of amino acids that have been considered as a characteristic signature [7].…”

“…In A . mellifera , members of the CPR, Tweedle, and Apidermin protein families have been most studied regarding developmental regulation of gene expression in tissues and organs [8, 9, 11, 14]. The other cuticular protein families found in the honeybee are known by the abbreviations CPF, CPFL, CPLCP, and CPAP3 [6, 9].…”

Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli

“…Deletion of DmTwld results in a decreased axial ratio and body shape alteration (Guan et al, 2006). In Apis mellifera, the expression of two Tweedle genes, AmelTwdl1 and AmelTwdl2, is determined by ecdysteroid titers, suggesting their potential roles in the formation of the cuticle (Soares et al, 2011). Four Tweedle genes are found in B. mori, though their functions have not yet been determined (Futahashi et al, 2008).…”