Genes cuticulares diferencialmente expressos durante eventos da metamorfose de Apis mellifera

SOARES, M. P. M. Microarray analysis of genes expressed in the context of Apis mellifera metamorphosis. Ph.D. Thesis – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2012. The insect cuticle is mainly composed of proteins that interact with chitin filaments to form a rigid structure that protects and shapes the organism. Insects grow through the periodic renewal of the cuticle, which is shed at each apolysis episode, and subsequently digested while the epidermis synthesizes the cuticle of the next stage. These molting events are coordinated by hormones, mainly ecdysteroids. The current work aimed to characterize differential gene expression in the integument (cuticle and underlying epidermis) during the ecdysteroid-regulated pupal-to-adult molt. Special attention was given to the structure and expression of genes encoding proteins and enzymes involved in cuticle formation and differentiation. To achieve these goals, we used thoracic integument of newly-ecdysed pupae (Pw), pupae in apolysis (Pp) and pharate adults (Pbl) in cDNA microarray analyses. The microarray analysis showed 761 and 1173 differentially expressed genes in the pharate adult integument (Pbl) in comparison to pupae (Pw) or pupae in apolysis (Pp), respectively. Gene Ontology terms for “Biological Process” and “Molecular Function” completely distinguished the integument of pharate adults (Pbl) from the integument of pupae (Pw) or pupae in apolysis (Pp). The microarray analysis discriminated 24 cuticular genes with a significant expression increase in the pharate adult integument. This was validated by real time RT-PCR analysis (qRT-PCR) for 23 of these genes (AmelCPR3, AmelCPR4, AmelCPR6, AmelCPR14, AmelCPR15, AmelCPR17, AmelCPR23, AmelCPR24, AmelCPR25, AmelCPR28, AmelCPR29, AmelCPR30, apd-1, apd-2, apd-3, CPLCP1, Am-C, Am-D, AmelTwdl1, AmelTwdl2, GB12449, GB12811 and GB11550), and by semiquantitative RT-PCR for Amlac2. In addition, the increased expression of other two cuticular genes (AmelCPR1 and AmelCPR2) was confirmed by qRT-PCR. These up-regulated cuticular genes in pharate adult integument apparently are involved in adult cuticle formation and differentiation, which occurs while the ecdysteroids titers decay, after reaching the peak that induces apolysis in the preceding phase (Pp). In contrast, two cuticular genes (AmelCPF1 e AmelCPR1) were confirmed by qRT-PCR analysis as negatively regulated in the integument of pharate adults compared to pupae, suggesting that they are specific to pupal cuticle. Therefore, these genes were inhibited by the increasing ecdysteroid levels that induce apolysis. Twenty one of the 24 cuticular genes differentially expressed in the microarrays encode proteins belonging to the CPF, CPR, Apidermin, CPLCP, Analogous to peritrofins and Tweedle families. The other three differentially expressed genes (GB12449, GB12811, GB11550) had not yet been assigned as cuticular genes. Two of them (GB12449 and GB12811) were sequenced, thus allowing prediction validation and gene structure characterization. In situ hybridization experiments using fluorescent probe (FISH) localized high expression of these genes in the pharate adult epidermis, strongly suggesting their involvement in the construction of the adult exoskeleton. This study is the first global gene expression analysis of the integument from a social hymenopteran species. The expression of genes in the integument was associated to the molting process and to the adult exoskeleton formation. This work contributes with new molecular data for a deeper understanding of A. mellifera metamorphosis.