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| Author(s): |
Aline Carolina Aleixo Silva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2012-08-13 |
| Examining board members: |
Zila Luz Paulino Simoes;
Angel Roberto Barchuk;
Marcia Maria Gentile Bitondi;
Anete Pedro Lourenço;
Francis de Morais Franco Nunes
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| Advisor: | Zila Luz Paulino Simoes |
| Abstract | |
In Apis mellifera, a eusocial bee, caste determination during larval development is regulated by differential nutrition. All female larvae are fed royal jelly until the third larval stage, when the workerdestined ones have their diet switched to a gland secretion mix, honey and pollen, Queen-destined larvae, however, are provisioned with a rich diet throughout development. Changes in diet after the third developmental stage modulate larval endocrine responses and different nutritional regimes trigger distinct patterns of gene expression. Thus, nutritional regulation of specific signaling pathways controls development of worker and queen phenotypes. Several proposed models of this process involve caste-specific regulation of hormonal and nutritional factors and/or molecular processes including Juvenile Hormone (JH), insulin/IGF and TOR (target of rapamycin) signaling pathways, differential methylation and the newly discovered protein royalactin, which facilitates queen development. Previous research has also suggested the stimulus of other factors in signaling pathways that acts towards workers development, and they possibly involves the participation of some genes like ultraspiracle (usp), cryptocephal (crc), and retinoid- and fatty acid-binding protein (RfaBp). Using diverse molecular approaches, we evaluate the role of these genes in caste differentiation. We used microarrays to characterize global differences in gene expression between queen and worker larvae. Functional analysis of significantly, differentially expressed genes identified fundamental biological processes including growth, reproduction, apoptosis, neurogenesis and JH degradation that are involved in caste differentiation. Based on these findings, we selected several candidate genes including juvenile hormone esterase (jhe), failed axon connections (fax), activating transcription factor-3 (atf-3), cathepsin-D (cath-D), and peptidoglycan recognition protein-SC2 (pgrp-sc2) for investigation. Notably, these genes were preferentially upregulated in workers. In a separate experiment, we monitored expression of usp, crc, RfaBp and jhe, in queen and worker larval during stages critical to caste determination. In general, workers expressed crc, RfaBp and jhe at higher levels than queens. For usp, distinct expression levels between worker- and queen-destined larvae were observed only at specific points between the 5th larval stage and pre-pupal phase. Additionally we used RNA interference (RNAi) to monitor the impact of decreased levels of select candidate genes on larval development. We found that even small modification of gene expression levels inhibited or triggered expression of other genes, and, in some cases, caused developmental alterations. Furthermore, microRNAs (miRNAs) are also important regulators of gene expression during development and we identified miRNAs that were predicted jhe regulators and assessed their levels. Results determined that some miRNAs including let-7, miR-2796 e miR-263b were strong candidates for jhe regulation because they were significantly and negatively correlated with target gene expression levels. Furthermore, manipulation of target gene expression levels altered expression of most predicted miRNAs. These results were confirmed by deep sequencing of RNAi samples treated with double-stranded RNA for jhe gene (dsjhe) and controls (with no treatment) which also identified other candidate jhe regulators, like miR-100, miR-306 and mi-13b. Taken together, these results suggest that worker development is regulated by complex interactions that involve usp, crc, RfaBp and jhe in addition to other molecules, including miRNAs. These molecular participants coordinate development at specific time points by regulating activity of gene networks and each other, producing the differential development of workers and queens in A. mellifera. (AU) | |
| FAPESP's process: | 08/01446-4 - Molecular determinants of workers development in Apis mellifera |
| Grantee: | Aline Carolina Aleixo Silva |
| Support Opportunities: | Scholarships in Brazil - Doctorate |